Organogenic transformation and regeneration

ABSTRACT

This invention provides methods of transforming organogenic plant cells and regenerating plants from transformed cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the national phase under 35 U.S.C. §371 ofInternational Application No. PCT/US2003/038912, which has anInternational filing date of Dec. 8, 2003 and which designated theUnited States of America, which is a continuation-in-part ofInternational Application No. PCT/US2003/038664, filed Dec. 5, 2003,which claims the benefit of U.S. Provisional Application No. 60/431,323,filed Dec. 6, 2002, which are each incorporated by reference in theirentirety for all purposes.

FIELD OF THE INVENTION

The present invention relates generally to plant biotechnology,providing methods of transforming and regenerating plants.

BACKGROUND OF THE INVENTION

It has become possible to transfer nucleic acids from a wide variety oforganisms to plants utilizing recombinant DNA technology. For example,this technology has provided a mechanism to improve various agronomicproperties including plant resistance to drought, temperature extremes,pests, diseases, and herbicides. However, the lack of efficient andreproducible transformation and regeneration from plant cell culturesystems for many agriculturally significant crops has been a substantialbarrier to the application of genetic engineering technology to plants.Moreover, there is a potential for the occurrence of somaclonalvariation when plants are regenerated adventitiously in vitro.Somaclonal variation is genetic variability commonly observed in plantsthat originate from, e.g., a callus intermediate. This geneticvariability is typically undesirable as the maintenance of the geneticintegrity of transformed plants is generally an objective.

SUMMARY OF THE INVENTION

The present invention provides methods for the transformation oforganogenic plant cells and for the regeneration of plants directly fromtransformed organogenic cells. The methods described herein aregenerally more efficient than many pre-existing plant transformation andregeneration methodologies. In addition, the methods of the presentinvention typically minimize the potential for the occurrence ofsomaclonal variation, as plants are regenerated from organogenic cellsor tissues rather than from calli or embryogenic cells. Nucleic acidsare introduced into cells using essentially any delivery technique,including Agrobacterium-mediated delivery, and can confer a wide rangeof desired agronomic properties. These and a variety of other featuresof the present invention will be apparent upon a complete review of thefollowing disclosure.

In one aspect, the invention provides a method of producing transformedplant cells. The method includes culturing at least one non-apicalmeristemic cell to produce one or more organogenic cells, andintroducing at least one nucleic acid segment into the organogenic cellsto produce one or more transformed organogenic cells. In certainembodiments, the method further includes generating at least one plantfrom the transformed organogenic cells.

In another aspect, the invention relates to a method of producingtransformed plant cells that includes culturing at least one meristemiccell to produce at least one shoot. In addition, the method alsoincludes culturing at least one explant from the shoot to produce one ormore organogenic cells. In certain embodiments, the explant comprisesone or more non-apical meristemic cells. The method also includesintroducing at least one nucleic acid segment into the organogenic cellsto produce one or more transformed organogenic cells. In someembodiments, the method further includes generating at least one plantfrom the transformed organogenic cells.

The meristemic cells (e.g., non-apical meristemic cells) utilized in themethods of the invention are optionally derived from various sources. Insome embodiments, for example, meristemic cells are derived frommonocotyledonous plants, whereas in other embodiments, meristemic cellsare derived from dicotyledonous plants. To further illustrate,meristemic cells utilized in the methods described herein are optionallyderived from plants selected from the genera: Ananas, Musa, Vitis,Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna,Citrus, Carica, Persea, Prunus, Syragrus, Theobroma, Coffea, Linum,Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis,Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum,Petunia, Digitalis, Majorana, Mangifera, Cichorium, Helianthus, Lactuca,Bromus, Asparagys, Antirrhinum, Heterocallis, Nemesia, Pelargonium,Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucurbita,Cucuinis, Browaalia, Lolium, Malus, Apium, Gossypium, Vicia, Lathyrus,Lupinus, Pachyrhizus, Wisteria, Stizolobium, Agrostis, Phleum, Dactylis,Sorgum, Setaria, Zea, Oryza, Triticum, Secale, Avena, Hordeum,Saccharum, Poa, Festuca, Stenotaphrum, Cynodon, Coix, Olyreae, Phareae,Glycine, Pisuin, Psidium, Passiflora, Cicer, Phaseolus, Lens, Arachis,or the like. In certain embodiments, meristemic cells comprise pineapplecells selected from, e.g., Smooth Cayenne cells, Red Spanish cells,Perolera cells, Pernambuco cells, Primavera cells, or the like.

Nucleic acid segments introduced into organogenic cells according to themethods of the present invention typically confer one or more desiredagronomic properties on the transformed cells and/or plants generatedtherefrom. In some embodiments, for example, nucleic acid segmentsconfer resistance to the transformed organogenic cells from insects,drought, nematodes, viral disease, bacterial disease, herbicides, and/orthe like. In certain embodiments, nucleic acid segments encodepolypeptides (e.g., plant polypeptides, bacterial polypeptides, etc.).Optionally, the polypeptide is artificially evolved. In certainembodiments, polypeptides are heterologous to the organogenic cells. Insome embodiments, polypeptides are homologous to at least one endogenouspolypeptide of the organogenic cells. To illustrate, nucleic acidsegments optionally comprise or encode, e.g., ACC synthases, ACCoxidases, malic enzymes, malic dehydrogenases, glucose oxidases,chitinases, defensins, expansins, hemicellulases, xyloglucantransglycosylases, apetala genes, leafy genes, knotted-related genes,homeobox genes, Etr-related genes, ribonucleases, and/or the like. Toillustrate further, polypeptides optionally comprise at least onecarotenoid biosynthetic polypeptide that is selected from, e.g., anisopentenyl diphosphate isomerase, a geranylgeranyl pyrophosphatesynthase, a phytoene synthase, a phytoene desaturase, a ζ-carotenedesaturase, a lycopene β-cyclase, a lycopene ε-cyclase, a β-carotenehydroxylase, an β-hydroxylase, and/or the like.

In some embodiments, nucleic acid segments stably integrate into thegenome of the transformed organogenic cells, whereas in otherembodiments, nucleic acid segments are only transiently present inorganogenic cells. Nucleic acid segments optionally include selectablemarkers, e.g., such that the introduction of the segments into cells canbe confirmed. In certain embodiments, nucleic acid segments are operablylinked to a constitutive promoter. In other embodiments, nucleic acidsegments are operably linked to an inducible promoter. In someembodiments, nucleic acid segments encode at least one polypeptidetranscription factor. In certain embodiments, nucleic acid segmentsencode at least one promoter and/or at least one enhancer, which nucleicacid segments homologously recombine with at least one promoter and/orat least one enhancer of at least one endogenous gene. In someembodiments, nucleic acid segments comprise sense nucleic acid segmentsthat correspond to at least a portion of at least one endogenous gene.In other embodiments, nucleic acid segments comprise at least one sensenucleic acid segment that corresponds to at least a portion of at leastone exogenous gene. Optionally, nucleic acid segments comprise at leastone antisense nucleic acid segment that corresponds to at least aportion of at least one endogenous gene.

Nucleic acids can be introduced into cells according to the methods ofthe invention using essentially any technique. In some embodiments, forexample, nucleic acid segments are introduced into the organogenic cellsusing Agrobacterium-mediated delivery. To further illustrate, nucleicacid segments are optionally introduced into the organogenic cells usingat least one nucleic acid delivery technique selected from, e.g.,pollen-mediated delivery, direct nucleic acid transfer to at least oneprotoplast of the organogenic cells, microprojectile bombardment,microinjection, macroinjection of inflorescence, whisker-mediatedimpregnation, laser perforation, ultrasonification, and/or the like.

DETAILED DESCRIPTION I. Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular embodiments.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting. Units, prefixes, and symbols are denoted in the formssuggested by the International System of Units (SI), unless specifiedotherwise. Numeric ranges are inclusive of the numbers defining therange. As used in this specification and the appended claims, thesingular forms “a”, “an” and “the” also include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a cell” also includes two or more cells (e.g., in the form of a tissue,etc.), and the like. Further, unless defined otherwise, all technicaland scientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which the inventionpertains. The terms defined below, and grammatical variants thereof, aremore fully defined by reference to the specification in its entirety.

The term “nucleic acid” encompasses any physical string of monomer unitsthat can be corresponded to a string of nucleotides, including a polymerof nucleotides (e.g., a typical DNA or RNA polymer), PNAs, modifiedoligonucleotides (e.g., oligonucleotides comprising nucleotides that arenot typical to biological RNA or DNA in solution, such as2′-O-methylated oligonucleotides), and/or the like. A nucleic acid canbe e.g., single-stranded or double-stranded. Unless otherwise indicated,a particular nucleic acid sequence of this invention encompassescomplementary sequences, in addition to the sequence explicitlyindicated. The term nucleic acid is used interchangeably with, e.g.,oligonucleotide, polynucleotide, gene, cDNA, RNAi, and mRNA encoded by agene.

A “nucleic acid sequence” refers to the order and identity of thenucleotides in a nucleic acid segment.

A “polynucleotide” is a polymer of nucleotides (A, C, T, U, G, etc. ornaturally occurring or artificial nucleotide analogues) or a characterstring representing a polymer of nucleotides, depending on context.Either the given nucleic acid or the complementary nucleic acid can bedetermined from any specified polynucleotide sequence.

Two nucleic acids “correspond” when they have the same sequence, or whenone nucleic acid is complementary to the other, or when one nucleic acidis a subsequence of the other, or when one sequence is derived, bynatural or artificial manipulation from the other.

The term “gene” is used broadly to refer to any segment of a nucleicacid associated with a biological function. Thus, genes include codingsequences and optionally, the regulatory sequences required for theirexpression. Genes also optionally include nonexpressed DNA or RNAsegments that, for example, form recognition sequences for otherproteins. Genes can be obtained from a variety of sources, includingcloning from a source of interest or synthesizing from known orpredicted sequence information, and may include sequences designed tohave desired parameters.

The term “nucleic acid segment” refers to a polynucleotide ortranscribable analog thereof, of at least 25 nucleotides in length,usually at least 100 nucleotides in length, generally at least 200nucleotides in length, typically at least 300 nucleotides in length,more typically at least 400 nucleotides in length, and most typically atleast 500 nucleotides in length. To illustrate, a nucleic acid segmentcan include a full-length gene (e.g., a gene that encodes a polypeptide,such as a carotenoid biosynthetic polypeptide or the like), or asubsequence of such a gene.

The term “T-DNA” refers to a nucleic acid segment that can be mobilizedand transferred from an Agrobacterium into a plant cell to therebyintroduce the nucleic acid segment into the plant cell.

The term “linked” generally refers to nucleic acid segments that arecontiguous with one another and, where necessary to join two amino acidcoding regions, contiguous and in the same reading frame. The term“linked” also encompasses nucleic acids that co-segregate with oneanother. Further, the term “operably linked” or “operatively linked”refers to a functional linkage between two or more nucleic acidsegments. For example, a promoter and a nucleic acid segment thatencodes a polypeptide are operably linked when the promoter sequenceinitiates and mediates transcription of the nucleic acid segment.

The term “expression” refers to the transcription and accumulation ofsense (mRNA) or antisense RNA derived from the nucleic acid segments ofthe invention. Expression may also refer to translation of mRNA into apolypeptide, e.g., a carotenoid biosynthetic polypeptide or the like. Incertain embodiments of the invention, for example, carotenoidbiosynthetic polypeptides are expressed in preselected plant storageorgans, such as roots, seeds, fruits, etc., leading to enhancedaccumulation of one or more carotenoids (e.g., naturally producedcarotenoids) in that plant storage organ. Accordingly, the term“fruit-specific expression” refers to the expression of, e.g.,introduced carotenoid biosynthetic polypeptides that is substantiallylimited to fruit tissues of the plants of the invention, e.g., so as toeffect an altered accumulation of carotenoid that is “substantiallyspecific to fruit tissues” of the transformed plants.

The term “promoter” refers to a recognition site on a DNA sequence orgroup of DNA sequences that provides an expression control element for agene and to which RNA polymerase specifically binds and initiates RNAsynthesis (transcription) of that gene. The term “constitutive promoter”refers to an unregulated promoter that allows for continualtranscription of an associated gene. The term “inducible promoter”refers to a regulated promoter that allows for transcription of anassociated gene in the presence of another substance or inducer, such asan extracellular molecule (e.g., a substrate of an enzyme that isencoded by the gene).

The term “selectable marker” includes reference to a gene whoseexpression allows one to identify cells that comprise the marker gene,such as a nucleic acid segment (e.g., a T-DNA) that includes the markergene in addition to an encoded polypeptide. To illustrate, a selectablemarker gene product may confer herbicide resistance on transformed cellssuch that upon exposing a population of cells to an effective amount ofthe herbicide, only those cells that have been transformed remainviable.

The term “sense nucleic acid segment” generally refers to a codingnucleic acid segment. In contrast, the term “antisense nucleic acidsegment” typically refers a complement of a sense nucleic acid segment.

The term “transcription factor” refers to any factor that controls theprocess of transcription (i.e., the making of an RNA copy of a DNAsegment). Usually it is an enzyme or other protein, a coenzyme, avitamin, or another organic molecule.

The term “enhancer” refers to a DNA sequence that positively influencesthe expression of a gene, even if the enhancer is positioned somedistance from that gene.

Two nucleic acids are “recombined” when sequences from each of the twonucleic acids are combined in a progeny nucleic acid.

A nucleic acid segment “stably integrates” into the genome of a plant orplant cell when it is non-transiently introduced into that genome. Forexample, a heterologous nucleic acid segment that is permanentlyincorporated into a plant chromosome is stably integrated into thegenome of the corresponding plant cell or plant.

The term “transformation” refers to the transfer or introduction of anucleic acid segment into a plant or plant cell, whether theintroduction results in genetically stable inheritance of the nucleicacid segment or only a transient presence of the nucleic acid segment inthe genome of the plant or plant cell. Plant cells or plants thatinclude the introduced nucleic acid segments are referred to as“transgenic,” “recombinant,” or “transformed” plant cells or plants.

A polynucleotide sequence, such as a nucleic acid segment, is“heterologous” to an organism, or a second polynucleotide sequence, ifit originates from another species, or, if from the same species, ismodified from its original or native form. For example, a promoteroperably linked to a heterologous coding sequence refers to a codingsequence from a species different from that from which the promoter wasderived, or, if from the same species, a coding sequence which isdifferent from a naturally occurring allelic variants.

Nucleic acids are “homologous” when they are derived, naturally orartificially, from a common ancestral sequence. Homology is ofteninferred from sequence similarity between two or more nucleic acids.This occurs naturally as two or more descendent sequences deviate from acommon ancestral sequence over time as the result of mutation andnatural selection. Artificially homologous sequences may be generated invarious ways. For example, a nucleic acid sequence can be synthesized denovo to yield a nucleic acid that differs in sequence from a selectedparental nucleic acid sequence. Artificial homology can also be createdby artificially recombining one nucleic acid sequence with another, asoccurs, e.g., during cloning or chemical mutagenesis, to produce ahomologous descendent nucleic acid. Artificial homology may also becreated using the redundancy of the genetic code to synthetically adjustsome or all of the coding sequences between otherwise dissimilar nucleicacids in such a way as to increase the frequency and length of highlysimilar stretches of nucleic acids while minimizing resulting changes inamino acid sequences to the encoded gene products. Preferably, suchartificial homology is directed to increasing the frequency of identicalstretches of sequence of at least three base pairs in length. Morepreferably, it is directed to increasing the frequency of identicalstretches of sequence of at least four base pairs in length. It isgenerally assumed that two nucleic acids have common ancestry when theydemonstrate sequence similarity. However, the exact level of sequencesimilarity necessary to establish homology varies in the art. Ingeneral, for purposes of this disclosure, two nucleic acid sequences aredeemed to be homologous when they share enough sequence identity topermit direct recombination to occur between the two sequences, that is,anywhere along the two sequences.

The term “encoding” refers to a polynucleotide sequence encoding one ormore amino acids. The term does not require a start or stop codon. Anamino acid sequence can be encoded in any reading frame provided by apolynucleotide sequence.

The term “vector” refers to an extra-chromosomal element that is capableof replication in a cell and/or to which other nucleic acid segments canbe operatively linked so as to bring about replication of thosesegments. Such elements may be autonomously replicating sequences,genome integrating sequences, phage or nucleotide sequences, linear orcircular, of single- or double-stranded DNA or RNA, derived from anysource, in which a number of nucleotide sequences have been joined orrecombined into a construct that is capable of introducing nucleic acidsegments (e.g., that include one or more promoters and one or more genesthat encode enzymes, etc.) along with appropriate 3′ untranslatedsequences into a cell. A plasmid is an exemplary vector. Vectors orvector systems (e.g., binary vectors systems, trinary vector systems, orthe like) can include elements in addition to, e.g., a gene that encodesa carotenoid biosynthetic enzyme, such as those which facilitatetransformation of a pineapple cell or plant, those that allow forenhanced expression of included genes (e.g., promoters) in transformedplants or plant cells, those that facilitate selection of transformedplants or plant cells (e.g., selectable markers, reporter genes, etc.),or the like. The term “expression vector” refers to an extra-chromosomalelement that is capable of regulating the expression of a gene (e.g., agene encoding a polypeptide) when operatively linked to the gene withinthe vector.

The term “polypeptide” refers to a polymer comprising two or more aminoacid residues (e.g., a peptide or a protein). The polymer canadditionally comprise non-amino acid elements such as labels, quenchers,blocking groups, or the like and can optionally comprise modificationssuch as glycosylation or the like. The amino acid residues of thepolypeptide can be natural or non-natural and can be unsubstituted,unmodified, substituted or modified.

The term “carotenoid biosynthetic polypeptide” refers to a biocatalystor enzyme that catalyzes at least one step in the carotenoidbiosynthetic pathway. Carotenoid biosynthetic polypeptides include,e.g., geranylgeranyl pyrophosphate synthases, isopentenyl diphosphateisomerases, phytoene synthases, phytoene desaturases, ζ-carotenedesaturases, lycopene β-cyclases, lycopene ε-cyclases, β-carotenehydroxylases, ε-hydroxylases, and the like.

A “polypeptide sequence” refers to the order and identity of the aminoacids in a polypeptide.

The term “artificially evolved polypeptide” refers to a polypeptidecreated using one or more diversity generating techniques. For example,artificially evolved polypeptides employed in the practice of thepresent invention are optionally produced by recombining (e.g., viarecursive recombination or the like) two or more nucleic acids encodingone or more parental polypeptides, or by mutating one or more nucleicacids that encode polypeptides (e.g., using site directed mutagenesis,cassette mutagenesis, random mutagenesis, recursive ensemblemutagenesis, or the like). A nucleic acid encoding a parentalpolypeptide includes a polynucleotide or gene that, through themechanisms of transcription and translation, produces an amino acidsequence corresponding to a parental polypeptide, e.g., annon-artificially evolved or naturally-occurring polypeptide. The term,“artificially evolved polypeptide” also embraces chimeric polypeptidesthat include identifiable component sequences (e.g., functional domains,etc.) derived from two or more parents. For example, artificiallyevolved enzymes employed in the practice of the present invention aretypically evolved, e.g., to yield products with greater efficiency thannaturally-occurring enzymes.

The term “endogenous” refers to a substance that is natively produced orsynthesized within an organism or system.

The term “exogenous” refers to materials originating from outside of theorganism or cell. It refers to nucleic acid molecules used in producingtransformed or transgenic host cells and plants. As used herein,exogenous is intended to refer to any nucleic acid that is introducedinto a recipient cell, regardless of whether a similar nucleic acid mayalready be present in such cell.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acid or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using asequence comparison algorithms or by visual inspection.

A region of “high sequence similarity” refers to a region that is 90% ormore identical to a second selected region when aligned for maximalcorrespondence (e.g., manually or, e.g., using the common program BLASTset to default parameters). A region of “low sequence similarity” is 30%or less identical, more preferably, 40% or less identical to a secondselected region, when aligned for maximal correspondence (e.g., manuallyor using BLAST set with default parameters).

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides, refers to two or more sequences or subsequencesthat have at least 60%, preferably 80%, most preferably 90-95%nucleotide or amino acid residue identity, when compared and aligned formaximum correspondence, as measured using, e.g., a sequence comparisonalgorithm or by visual inspection. Preferably, the substantial identityexists over a region of the sequences that is at least about 50 residuesin length, more preferably over a region of at least about 100 residues,and most preferably the sequences are substantially identical over atleast about 150 residues. In some embodiments, the sequences aresubstantially identical over the entire length of the coding regions.

A “subsequence” or “fragment” is any portion of an entire sequence ofnucleic acids or amino acids.

The term “genome” refers to the chromosomal nucleic acids of anorganism. As used herein, genome also includes the plastid genome (e.g.,chloroplast genome, etc.).

The term “Agrobacterium” refers to species, subspecies, or strains ofthe bacterium Agrobacterium that are able to mobilize and selectivelytransfer T-DNA into a plant cell. For example, the Agrobacterium isoptionally Agrobacterium rhizogenes, but more typically is Agrobacteriumtumefaciens.

The term “callus” refers to an undifferentiated proliferating mass ofcells or tissue.

The term “embryogenic cell” refers to a cell from embryogenic tissue orembryogenic callus.

The term “embryogenic callus” refers to tissue or cells that areundifferentiated and without significant structure but with thepotential to form a more differentiated tissue (e.g., embryogenictissue) that can produce embryos and germinate into plants.

The term “embryogenic tissue” refers to tissue having organizedstructures that include immature somatic embryos. Immature somaticembryos can be matured by culturing them on a maturation medium. Maturesomatic embryos can develop into plants upon transfer to a germinationmedium. A mature somatic embryo is a structure ultimately derived fromsomatic cells that resembles a zygotic embryo morphologically anddevelopmentally, and that is capable of germinating into a plantlet withboth root and shoot poles, when transferred to a suitable growth medium.A “somatic cell” is a cell of a multicellular organism other thangametes.

The term “organogenic cell” refers to a cell from organogenic tissue.

The term “organogenic callus” refers to tissue having an irregular massof relatively undifferentiated cells, which can arise from, e.g., asingle organogenic cell in tissue culture.

The term “organogenic tissue” refers to tissue that is capable of beinginduced to undergo organogenesis, that is, to form a plant organ such asa shoot, which can then be induced to develop roots to produce acomplete plant.

The term “regenerating” or “generating” refers to the formation of aplant that includes a rooted shoot.

The term “effective amount” refers to an amount sufficient to achieve adesired result such as the production of a callus or tissue that isembryogenic or organogenic.

The term “dicot” or “dicotyledonous” refers to plants that produce anembryo with two cotyledons. Exemplary dicots include cotton, soybean,and peanut.

The term “monocot” or “monocotyledonous” refers to plants having asingle cotyledon. Exemplary monocots include pineapple, maize, rice,wheat, oat, and barley.

The term “explant” refers to living tissue removed from an organism andplaced in a medium for tissue culture.

The term “meristem” refers to a formative plant tissue that comprisescells capable of dividing and giving rise to similar cells or to cellsthat differentiate to produce tissues and organs.

The term “meristemic cell” refers to a cell from a plant meristem.

The term “non-apical meristemic cell” refers to a meristemic cell thatis not from apical meristem, but can be from lateral or axilliarymeristems and from cells that upon culture of a non-apical meristemexplant have become meristematic.

The term “selecting” refers to a process in which one or more plants orplant cells are identified as having one or more properties of interest,e.g., a selectable marker, enhanced nematode resistance, increased ordecreased carotenoid levels, altered coloration, etc. For example, aselection process can include placing organisms under conditions wherethe growth of those with a particular genotype will be favored. Tofurther illustrate, one can screen a population to determine one or moreproperties of one or more members of the population. If one or moremembers of the population is/are identified as possessing a property ofinterest, it is selected. Selection can include the isolation of amember of a population, but this is not necessary. In addition,selection and screening can be, and often are, simultaneous.

The term “screening” refers to a process for separating a populationinto different groups. Screening processes typically include determiningone or more properties of one or more plants or plant cells. Forexample, typical screening processes include those in which one or moreproperties of one or more members of one or more populations is/aredetermined.

II. Methods of Producing Transformed Plant Cells, Cell Culture, andExplant Sources

The present invention relates generally to methods of geneticallytransforming cells and plants. The methods of the invention aretypically more efficient than many pre-existing plant transformation andregeneration techniques. Exemplary plant traits that can be modifiedusing the methods described herein include fruit quality (e.g.,sweetness, acidity, texture, condition, color (e.g., shell color or thelike), etc.), fruit ripening characteristics, nutritional value (e.g.,modified carotenoid levels, etc.), among many other traits typically ofinterest to consumers. Optionally, other agronomic traits such as,improved flowering control, improved resistance to drought, improvedresistance to bacterial diseases, improved resistance to viral diseases,and/or improved resistance to insects, nematodes, and herbicides arealso engineered into the cells and plants of the invention. In certainembodiments, the methods of the invention include the use of suitableexplant material, which is genetically transformed by contacting theexplant material with Agrobacterium cells. The Agrobacterium cellsmediate the transfer of nucleic acid segments, e.g., that encodepolypeptides, into plant cells. Other techniques for delivering nucleicacid segments into cells or plants are also optionally utilized. Theinvention also provides culture media suitable for the steps of inducingthe formation of organogenic cells for co-cultivation with Agrobacteriumcells.

In overview, one aspect of the invention provides a method of producingtransformed plant cells that includes culturing at least one non-apicalmeristemic cell to produce one or more organogenic cells, andintroducing at least one nucleic acid segment into the organogenic cellsto produce one or more transformed organogenic cells. In another aspect,the invention relates to a method of producing transformed plant cellsthat includes culturing at least one meristemic cell to produce at leastone shoot. In addition, the method also includes culturing at least oneexplant from the shoot to produce one or more organogenic cells, andintroducing at least one nucleic acid segment into the organogenic cellsto produce one or more transformed organogenic cells. The methodsdescribed herein also typically further include generating at least oneplant from the transformed organogenic cells.

The methods of the invention include culturing meristemic cells (e.g.,non-apical meristemic cells). In some embodiments, for example,meristemic cells are derived from monocotyledonous plants, whereas inothers, meristemic cells are derived from dicotyledonous plants. Tofurther illustrate, meristemic cells are optionally derived from plantsselected from the genera: Ananas, Musa, Vitis, Fragaria, Lotus,Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Carica,Persea, Prunus, Syragrus, Theobroma, Coffea, Linum, Geranium, Manihot,Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum,Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia,Digitalis, Majorana, Mangifera, Cichorium, Helianthus, Lactuca, Bromus,Asparagus, Antirrhinum, Heterocallis, Nemesia, Pelargonium, Panicum,Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucurbita, Cucumis,Browaalia, Lolium, Malus, Apium, Gossypium, Vicia, Lathyrus, Lupinus,Pachyrhizus, Wisteria, Stizolobium, Agrostis, Phleum, Dactylis, Sorguin,Setaria, Zea, Oryza, Triticum, Secale, Avena, Hordeum, Saccharum, Poa,Festuca, Stenotaphrum, Cynodon, Coix, Olyreae, Phareae, Glycine, Pisum,Psidium, Passiflora, Cicer, Phaseolus, Lens, Arachis, or the like.

To further exemplify, essentially any pineapple variety may betransformed according to the methods described herein. For example,pineapple merstemic cells are optionally obtained from varieties thatare typically used for human consumption, including those of the SmoothCayenne group, the Spanish group (e.g., Red Spanish), the Peroleragroup, the Pernambuco group, and the Primavera group. The most importantvariety for use in the production of canned pineapple, other processedpineapple products, and fresh pineapple is typically Smooth Cayenne.Within many of these varieties there are a large number of clones whichhave been established in different geographical areas, and which areadapted to production in those locations. Among the Smooth Cayenneclones are the Champaka clones which have been used extensively forproduction of canned and fresh pineapple.

In certain embodiments, rapidly growing shoot cultures are produced invitro, then explants such as leaf pieces, petioles, cotyledons, stemsections, peduncles, etc. are cultured to produce meristemic ororganogenic cells, e.g., in pretreatment processes that prepare celland/or tissues for cocultivation with Agrobacterium cells. Initialexplants can be any meristemic region of a plant, including either themain or axillary meristems (apices) of the plant prior to, e.g., flowerformation, and the main or axillary meristems of, e.g., the crown of thefruit in pineapple or another plant. These regions can be excised fromthe plant and sterilized by standard methods as described herein andwell known to those of ordinary skill in the art to establish sterilecultures in an artificial medium. Such cultures can be maintained for anextended period of time (e.g., weeks, months or years) by a series ofpropagation steps. Suitable media for establishment and maintenance ofin vitro shoot cultures are described in, e.g., DeWald et al. (1988)Plant Cell Reports, 7:535-537; Wakasa et al. (1978) Japan J Breed28:113-121; Mathews and Rangan (1981) Scientia Hort 14:227-234;Srinivasa et al. (1981) Scientia Hort 15: 23S-238; Fitchet (1990) ActaHort 275:267-274; Bordoloi and Sarma (1993) J Assam Science Society35:41-45; and Firoozabady and Moy (2004) “Regeneration of pineappleplants via somatic embryogenesis and organogenesis,” In Vitro Cellularand Developmental Biology—Plant 40(1). Additional details relating toculturing plant cells, including pretreatment processes, are providedbelow in the examples.

One skilled in the art will recognize that many different types oforganogenic cells can be used as target cells for the delivery ofnucleic acid segments and selection of transformation events. Forexample, nucleic acid segments can be delivered to the cells of theleaf, leaf base, or stem sections as they undergo organogenesis. Inparticular, nucleic acid segments can be delivered to organogenic cellsafter the organogenic material has been maintained and proliferated invitro for a selected period of time. In certain embodiments, as referredto above, the plant cells which are the target for nucleic acid segmentdelivery are first obtained from the basal portion of leaves (i.e., leafbase) or sections of the stem of shoots grown in vitro, and proliferatedin culture prior to the nucleic acid segment delivery step. As usedherein, the term “leaf base” refers to that portion of the leaf that isconnected to the stem of a shoot.

Additional details relating to cell culture are described in, e.g.,Published International Application No. WO 01/33943, entitled “A METHODOF PLANT TRANSFORMATION,” by Graham et al., which published May 17,2001, U.S. Pat. No. 5,908,771, entitled “METHOD FOR REGENERATION OFSALVIA SPECIES,” which issued Jun. 1, 1999 to Liu et al., U.S. Pat. No.6,242,257, entitled “TISSUE CULTURE PROCESS FOR PRODUCING A LARGE NUMBEROF VIABLE COTTON PLANTS IN VITRO,” which issued Jun. 5, 2001 to Tuli etal., Croy (Ed.) Plant Molecular Biology Labfax, Bios ScientificPublishers Ltd. (1993), Jones (Ed.) Plant Transfer and ExpressionProtocols, Humana Press (1995), and in the references cited therein.

Nucleic acid segments can be introduced into cells in a number ofart-recognized ways. In overview, suitable methods of transforming plantcells include microinjection (Crossway et al. (1986) BioTechniques4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci.USA 83:5602-5606, Agrobacterium-mediated transformation (Hinchee et al.(1988) Biotechnology 6:915-921), ballistic particle acceleration ormicroprojectile bombardment (Sanford et al. U.S. Pat. No. 4,945,050; andMcCabe et al. (1988) Biotechnology 6:923-926), pollen-mediated delivery(Zhou et al. (1983) Methods Enzymol. 101:433, De Wet et al. (1985) inThe Experimental Manipulation of Ovule Tissues, Chapman et al. (Eds.),Longman, p. 197; Hess (1987) Intern. Rev. Cytol. 107:367; and Luo et al.(1989) Plant Mol. Biol. Rep. 7:69), direct nucleic acid transfer toprotoplasts of pineapple cells (Caboche et al. (1984) Comptes RendusAcad. Sci. 299, series 3:663), microinjection (Crossway et al. (1986)Mol. Gen. Genet. 202:179 and Reich et al. (1986) Bio/technol. 4:1001),macroinjection of inflorescence (De la Pena et al. (1987) Nature325:274), whisker-mediated impregnation (Dunahay (1993) Biotechniques15:452-460 and Frame et al. (1994) The Plant Journal 6:941-948), laserperforation (Weber (1988) Naturwissenschaften 75:35), andultrasonification (Zhang et al. (1991) Bio/technol. 9:994).

To further illustrate, Agrobacterium-mediated transfer is a widelyapplicable system for introducing genes into plant cells because thenucleic acid segments can be introduced into whole plant tissues,thereby bypassing the need for regeneration of an intact plant from aprotoplast. The use of Agrobacterium-mediated expression vectors tointroduce DNA into plant cells is well known in the art. See, e.g., themethods described by Fraley et al. (1985) Biotechnology, 3:629 andRogers et al. (1987) Methods in Enzymology 153:253-277. Further, theintegration of T-DNA is a relatively precise process resulting in fewrearrangements. The region of DNA to be transferred is defined by theborder sequences, and intervening DNA or nucleic acid segment is usuallyinserted into the plant genome as described by Spielmann et al. (1986)Mol. Gen. Genet., 205:34 and Jorgensen et al. (1987) Mol. Gen. Genet.207:471.

Many Agrobacterium transformation vectors are capable of replication inE. coli as well as Agrobacterium, allowing for convenient manipulationsas described by Klee et al., in Plant DNA Infectious Agents, Hohn andSchell, (Eds.), Springer-Verlag (1985) pp. 179-203.

Moreover, recent technological advances in vectors forAgrobacterium-mediated gene transfer have improved the arrangement ofgenes and restriction sites in the vectors to facilitate construction ofvectors capable of expressing various polypeptide coding genes. Forexample, the vectors described by Rogers et al. (1987) Methods inEnzymology, 153:253, have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes. Suitablevectors are described in greater detail herein.

In certain embodiments of the invention, heterologous nucleic acidsegments are introduced using Agrobacterium strains carrying theexogenous DNA in a T-DNA element. The recombinant T-DNA element caneither be part of a Ti-plasmid that contains the virulence functionsnecessary for DNA delivery from Agrobacterium cells to plant cells, orthe T-DNA element can be present on a plasmid distinct from anotherplasmid carrying the virulence functions (referred to as binaryvectors). A variety of these binary vectors, capable of replication inboth E. coli and Agrobacterium, are described in the references citedabove. In certain methods of co-cultivation, Agrobacterium is grown to aconcentration of 2-7×10⁸ cells/ml and is diluted to 1-6×10⁸ cells/ml,preferably 2-5×10⁸ cells/ml before co-cultivation. Agrobacterium istypically co-cultivated with plant tissues for about 1-7 days, and moretypically for about 2-3 days with, e.g., certain plant tissues, such aspineapple tissues.

Suitable Agrobacterium strains include Agrobacterium tumefaciens andAgrobacterium rhizogenes. While wild-type strains may be used,“disarmed” derivatives of both species, in which the tumor-inducingsequences of the Ti plasmid have been removed, are preferred. SuitableAgrobacterium tumefaciens strains include, e.g., EHA101, as described byHood et al. ((1986) J. Bacteriol., 168:1291-1301), LBA4404, as describedby Hoekema et al. ((1983) Nature, 303:179-80), and C58(pMP90), asdescribed by Koncz and Schell ((1986) Mol. Gen. Genet., 204:383-96). Apreferred Agrobacterium rhizogenes strain is 15834, as described byBirot et al. (Biochem, 25: 323-35).

The organogenic cells and tissues and the Agrobacterium cells carryingthe nucleic acid segment are co-cultivated in a suitable co-cultivationmedium to allow transfer of the T-DNA to plant cells. After theAgrobacterium strain carrying the nucleic acid segment has beenprepared, it is usually cultured prior to incubation with the cells.Agrobacterium can be cultured on solid or liquid media according tomethods well known to those of skill in the art. See, e.g., U.S. Pat.No. 5,262,316.

As additional options, transformation of plant protoplasts can beachieved using methods based on calcium phosphate precipitation,polyethylene glycol treatment, electroporation, and combinations ofthese treatments. See, e.g., Potrykus et al. (1985) Mol. Gen. Genet.,199:183; Lorz et al. (1985) Mol. Gen. Genet., 199:178; Fromm et al.(1986) Nature, 319:791; Uchimiya et al. (1986) Mol. Gen. Genet.,204:204; Callis et al. (1987) Genes and Development, 1:1183; Marcotte etal. (1988) Nature, 335:454; Wang et al. (1992) Bio/Technology10:691-696; and Fennell et al. (1992) Plant Cell Reports, 11:567-570.

To transform plant species that cannot be successfully regenerated fromprotoplasts, other ways to introduce nucleic acid segments into intactcells or tissues can be utilized. For example, “particle gun” orhigh-velocity microprojectile technology can be utilized. Using suchtechnology, nucleic acid segments are carried through the cell wall andinto the cytoplasm on the surface of small metal particles as describedin Klein et al. (1987) Nature, 327:70; Klein et al. (1988) Proc. Natl.Acad. Sci. U.S.A., 85:8502; and McCabe et al. (1988) Biotechnology,6:923; and Vasil et al. (1992) Bio/Technology, 9:667-674. The metalparticles penetrate through several layers of cells and thus allow forthe transformation of cells within tissue explants. Transformation oftissue explants eliminates the need for passage through a protoplaststage and thus speeds the production of transgenic plants.

Nucleic acid segments are also optionally introduced into plants inperforming the methods of the invention by direct nucleic acid transferinto pollen as described by Zhou et al. (1983) Methods in Enzymology101:433; Hess (1987) Intern Rev. Cytol. 107:367; Luo et al. (1988) PlantMol. Biol. Reporter 6:165. Expression of polypeptide coding genes can beobtained by injection of the nucleic acid segment into reproductiveorgans of a plant as described by Pena et al. (1987) Nature 325:274.

Alternatively, a plant plastid can be transformed directly in performingthe methods described herein. Stable transformation of chloroplasts hasbeen reported in higher plants, see, e.g., Svab et al. (1990) Proc.Nat'l. Acad. Sci. USA 87:8526-8530; Svab et al. (1993) Proc. Nat'l Acad.Sci. USA 90:913-917; Staub et al. (1993) Embo J. 12:601-606. The methodutilizes particle gun delivery of nucleic acid segments containing aselectable marker and targeting of the nucleic acid to the plastidgenome through homologous recombination. In such methods, plastid geneexpression can be accomplished by use of a plastid gene promoter or bytrans-activation of a silent plastid-borne transgene positioned forexpression from a selective promoter sequence such as that recognized byT7 RNA polymerase. The silent plastid gene is activated by expression ofthe specific RNA polymerase from a nuclear expression construct andtargeting of the polymerase to the plastid by use of a transit peptide.Tissue-specific expression may be obtained in such a method by use of anuclear-encoded and plastid-directed specific RNA polymerase expressedfrom a suitable plant tissue specific promoter. Such a system has beenreported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA91:7301-7305.

After delivery of nucleic acid segments, organogenic cells are typicallytransferred to media that include a selective agent (e.g., an herbicideor the like) that is capable of preventing the growth of cells that havenot received a gene (e.g., a selectable marker) whose expression productis capable of preventing the action of the selective agent to therebyselect for transformed plant cells. In certain embodiments, for example,tissues are exposed to sublethal levels of selective agents for about2-12 weeks, and then to lethal levels of selective agents for about 4-30weeks in a step-wise selection process. Selectable markers are describedfurther herein. In certain embodiments, organogenic cells aretransferred to a recovery medium that comprises counter-selective agents(e.g., antibiotics, etc.), e.g., to kill Agrobacterium cells for aperiod of about 1-15 days, e.g., prior to or concurrently with beingtransferred to media comprising a selective agent. After a period ofculture, organogenic cells that continue to grow normally are separatedfrom cells whose growth has been slowed or terminated.

The regeneration of plants from either single plant protoplasts orvarious explants is well known in the art. See, e.g., Weissbach et al.(Eds.), Methods for Plant Molecular Biology, Academic Press, Inc.(1988). In certain embodiments of the invention, the regeneration andgrowth process includes the steps of selecting transformed cells andshoots, rooting the transformed shoots, and growing the plantlets insoil. To illustrate, the regeneration of plants containing a geneintroduced by Agrobacterium from leaf explants can be achieved asdescribed by Horsch et al. (1985) Science, 227:1229-1231. In thisprocedure, transformants are grown in the presence of a selection agentand in a medium that induces the regeneration of shoots in the plantspecies being transformed as described by Fraley et al. (1983) Proc.Natl. Acad. Sci. U.S.A., 80:4803. This procedure typically producesshoots within two to four weeks and these transformed shoots are thentransferred to an appropriate root-inducing medium containing theselective agent and an antibiotic to prevent bacterial growth. Inpineapples, for example, leaf bases may be used to produce organogenicmaterials (Firoozabady and Moy (2004) “Regeneration of pineapple plantsvia somatic embryogenesis and organogenesis,” In Vitro Cellular andDevelopmental Biology—Plant 40(1)), and then these materials may beexposed to Agrobacterium to produce, upon selection, transgenicorganogenic materials. These materials then are induced to produceshoots and complete plants. Typically, transformed shoots that rooted inthe presence of the selective agent to form plantlets are thentransplanted to soil or other media to allow the production ofadditional roots.

Additional details relating to plant regeneration, micropropagation, andother aspects that are adapted for use in the methods of the presentinvention are provided in, e.g., U.S. Pat. No. 5,952,543 and WO 01/33943(referenced above), U.S. Pat. Nos. 5,591,616, 6,037,522, European Pat.Application Nos. 604662 (A1) and 672752 (A1), and WO 01/12828. See also,Kyte et al., Plants from Test Tubes: An Introduction toMicropropagation, Timber Press, Inc. (1996), Hudson et al., Hartmann andKester's Plant Propagation: Principles and Practices 7^(th) Ed., PearsonEducation (2001), Bajaj (Ed.) High-Tech and Micropropagation I,Springer-Verlag New York, Inc. (1992), Jain, In Vitro Haploid Productionin Higher Plants, Kluwer Academic Publishers (1996), and Debergh et al.(Eds.), Micropropagation: Technology and Application, Kluwer AcademicPublishers (1991).

Optionally, polypeptides produced in transformed cells or plants can berecovered and purified from transformed cell cultures or transformedplant tissues (e.g., fruit tissues or the like) by any of a number ofmethods well known in the art, including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography, and lectinchromatography. In some cases the protein will need to be refolded torecover a functional product. In addition to the references noted supra,a variety of purification methods are well known in the art, including,e.g., those set forth in Sandana, Bioseparation of Proteins, AcademicPress, Inc. (1997); and Bollag et al., Protein Methods 2nd Ed.,Wiley-Liss, NY (1996); Walker, The Protein Protocols Handbook, HumanaPress, NJ (1996); Harris and Angal, Protein Purification Applications: APractical Approach, IRL Press (1990); Scopes, Protein Purification:Principles and Practice, 3rd Ed., Springer Verlag (1993); and Janson etal., Protein Purification: Principles, High Resolution Methods andApplications, 2^(nd) Ed., Wiley-VCH (1998).

III. Nucleic Acid Segment Selection

A wide variety of inorganic or organic molecules are optionallyintroduced into cells using the methods described herein to modifytraits as desired. In some embodiments, for example, nucleic acidsegments that encode polypeptides (e.g., plant polypeptides, bacterialpolypeptides, etc.) are introduced. In certain of these embodiments, thepolypeptide is heterologous to the organogenic cells. In someembodiments, the polypeptide is homologous to an endogenous polypeptideof the organogenic cells. Essentially any nucleic acid segment isoptionally utilized to transform the organogenic cells according to themethods described herein. Accordingly, no attempt is made to identifyall of the known nucleic acids that can be utilized. However, toillustrate, some carotenogenesis-related nucleic acid segments that areoptionally introduced into cells or plants typically encode, e.g.,isopentenyl diphosphate isomerases, geranylgeranyl pyrophosphatesynthases, phytoene synthases, phytoene desaturases, ζ-carotenedesaturases, lycopene β-cyclases, lycopene ε-cyclases, β-carotenehydroxylases, ε-hydroxylases, and/or the like. Additional detailsrelating to these carotenogenesis-related nucleic acid segments aredescribed in, e.g., PCT/US03/38664, entitled “TRANSGENIC PINEAPPLEPLANTS WITH MODIFIED CAROTENOID LEVELS AND METHODS OF THEIR PRODUCTION,”filed Dec. 5, 2003 by Young et al., which is incorporated by reference.Other exemplary nucleic acid segments optionally comprise or encode,e.g., an ACC synthase, an ACC oxidase, a malic enzyme, a malicdehydrogenase, a glucose oxidase, a chitinase, a defensin, an expansin,a hemicellulase, a xyloglucan transglycosylase, an apetala gene, a leafygene, a knotted-related gene, a homeobox gene, an Etr-related gene, aribonuclease, and/or the like.

IV. Artificially Evolved Polypeptides

In certain embodiments of the invention, artificially evolvedpolypeptides are used to modulate traits in transgenic cells and plants.For example, any of the exemplary target carotenoid biosyntheticpolypeptides described above can be artificially evolved to acquiredesired traits or properties, such as increased catalytic efficiency,increased substrate specificity, and/or the like. A variety ofartificial diversity generating procedures are available and describedin the art, which can be used to produce artificially evolvedpolypeptides. These procedures can be used separately or in combinationto produce variants of a nucleic acid, as well as variants of proteinsencoded by the nucleic acid variants. Individually and collectively,these procedures provide robust, widely applicable ways of engineeringor rapidly evolving individual nucleic acids and proteins, or evenentire biochemical pathways or selected portions of such pathways. Theproducts of these procedures can be used in the transformation methodsof the invention.

In particular, the result of any of the diversity generating proceduresdescribed herein or otherwise known in the art can be the generation ofone or more nucleic acids that are typically selected or screened fornucleic acids encoding enzymes with or which confer desirableproperties, such as increased catalytic efficiency, etc. This caninclude identifying any activity that can be detected, for example, inan automated or automatable format, by any of the assays known in theart. A variety of related (or even unrelated) properties can beevaluated, in serial or in parallel, at the discretion of thepractitioner.

Artificially evolved polypeptides that are optionally used in themethods of the present invention can be derived using many differenttechniques. To illustrate, chimeric enzymes including identifiablecomponents (e.g., protein domains) derived from two or more parentalsequences can be utilized. For example, domains in different phytoenesynthases can be identified and selected for inclusion in a chimericprogeny phytoene synthase. Various sequence comparison algorithms andother tools that are useful for chimeric enzyme design are describedfurther below.

Artificially evolved enzymes can additionally be developed using variousmutagenic methods, such as cassette mutagenesis, site-directedmutagenesis (see, e.g., Botstein & Shortle (1985) “Strategies andapplications of in vitro mutagenesis” Science 229:1193-1201; Carter(1986) “Site-directed mutagenesis” Biochem. J. 237:1-7; and Kunkel(1987) “The efficiency of oligonucleotide directed mutagenesis” inNucleic Acids & Molecular Biology (Eckstein, F. and Lilley, D. M. J.eds., Springer Verlag, Berlin)), chemical mutagenesis, error-prone PCR,site-saturation mutagenesis, recursive ensemble mutagenesis, and thelike. To illustrate, error-prone PCR can be used to generate nucleicacid variants. Using error-prone PCR, for example, PCR is performedunder conditions where the copying fidelity of the DNA polymerase islow, such that a high rate of point mutations is obtained along theentire length of the PCR product. Examples of such techniques aredescribed further in, e.g., in Leung et al. (1989) Technique 1:11-15 andCaldwell et al. (1992) PCR Methods Applic. 2:28-33. To furtherillustrate an exemplary mutagenic technique, cassette mutagenesis isoptionally used in a process that replaces a small region of a doublestranded DNA molecule with, e.g., a synthetic oligonucleotide cassettethat differs from the native sequence. The synthetic oligonucleotide cancontain, e.g., completely and/or partially randomized nativesequence(s). Additional details relating to cassette mutagenesis aredescribed in, e.g., Wells et al. (1985) “Cassette mutagenesis: anefficient method for generation of multiple mutations at defined sites”Gene 34:315-323. Another exemplary method of creating moleculardiversity mutagenically is a recursive ensemble mutagenesis process inwhich an algorithm for protein mutagenesis is used to produce diversepopulations of phenotypically related mutants, members of which differin amino acid sequence. This method uses a feedback mechanism to monitorsuccessive rounds of combinatorial cassette mutagenesis. Examples ofthis approach are found in Arkin et al. (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815. The mutagenic techniques referred to above areprovided simply to illustrate certain procedures that are optionallyused to produce diversity in nucleic acids that encode polypeptides.Many other approaches to creating diversity via mutagenesis arewell-known and are also suitable for the methods of the presentinvention.

Nucleic acids that encode polypeptides, such as those described above,are also optionally used as substrates for a variety of recombinationreactions, such as a recursive sequence recombination protocol. Many ofthese techniques are described in various publications including, e.g.,Chang et al. (1999) “Evolution of a cytokine using DNA family shuffling”Nature Biotechnology 17:793-797, Crameri et al. (1998) “DNA shuffling ofa family of genes from diverse species accelerates directed evolution”Nature 391:288-291, Crameri et al. (1997) “Molecular evolution of anarsenate detoxification pathway by DNA shuffling,” Nature Biotechnology15:436-438, Crameri et al. (1996) “Improved green fluorescent protein bymolecular evolution using DNA shuffling” Nature Biotechnology14:315-319, Stemmer (1995) “Searching Sequence Space” Bio/Technology13:549-553, and Stemmer (1994) “Rapid evolution of a protein in vitro byDNA shuffling” Nature 370:389-391.

Many of the diversity generating procedures described above (e.g.,chimeric enzyme design and synthesis, recursive sequence recombination,etc.), include determining levels of homology among starting sequences.For example, in the processes of sequence comparison and homologydetermination, one sequence, e.g., one fragment or subsequence of a genesequence to be recombined, can be used as a reference against whichother test nucleic acid sequences are compared. This comparison can beaccomplished with the aid of a sequence comparison algorithm or byvisual inspection. When an algorithm is employed, test and referencesequences are input into a computer, subsequence coordinates aredesignated, as necessary, and sequence algorithm program parameters arespecified. The algorithm then calculates the percent sequence identityfor the test nucleic acid sequence(s) relative to the referencesequence, based on the specified program parameters.

For purposes of the present invention, suitable sequence comparisons canbe executed, e.g., by the local homology algorithm of Smith & Waterman(1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm ofNeedleman & Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson & Lipman (1988) Proc. Nat'l. Acad. Sci. USA85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visualinspection. See generally, Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 2001). Another example search algorithm that is suitable fordetermining percent sequence identity and sequence similarity is theBasic Local Alignment Search Tool (BLAST) algorithm, which is describedin, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410. Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information on the world wide web atncbi.nlm.nih.gov.

Kits for mutagenesis, library construction and other diversitygeneration methods are commercially available. For example, kits areavailable from, e.g., Stratagene (e.g., QuickChange™ site-directedmutagenesis kit, and Chameleon™ double-stranded, site-directedmutagenesis kit), Bio/Can Scientific, Bio-Rad (e.g., using the Kunkelmethod referred to above), Boehringer Mannheim Corp., ClonetechLaboratories, DNA Technologies, Epicentre Technologies (e.g., 5 prime 3prime kit), Genpak Inc., Lemargo Inc., Life Technologies (Gibco BRL),New England Biolabs, Pharmacia Biotech, Promega Corp., QuantumBiotechnologies, Amersham International plc, and Anglian BiotechnologyLtd.

V. Nucleic Acid Segment Preparation

Nucleic acid segments used in the methods of the invention, such asthose described above, can be prepared using various methods orcombinations thereof, including certain DNA synthetic techniques, DNAamplification, nuclease digestion, etc. Searchable sequence informationthat is available from nucleic acid databases can be utilized duringnucleic acid segment and vector selection and/or design processes.Genbank®, Entrez®, EMBL, DDBJ, GSDB, NDB and the NCBI are examples ofpublic database/search services that can be accessed. These databasesare generally available via the internet or on a contract basis from avariety of companies specializing in genomic information generationand/or storage. These and other helpful resources are readily availableand known to those of skill.

The sequence of a polynucleotide to be used in any of the methods of thepresent invention can also be readily determined using techniqueswell-known to those of skill, including Maxam-Gilbert, Sanger Dideoxy,and Sequencing by Hybridization methods. For general descriptions ofthese processes consult, e.g., Stryer, Biochemistry, 4^(th) Ed., W.H.Freeman and Company (1995) and Lewin, Genes VI, Oxford University Press(1997). See also, Maxam and Gilbert (1977) “A New Method for SequencingDNA,” Proc. Natl. Acad. Sci. 74:560-564, Sanger et al. (1977) “DNASequencing with Chain-Terminating Inhibitors,” Proc. Natl. Acad. Sci.74:5463-5467, Hunkapiller et al. (1991) “Large-Scale and Automated DNASequence Determination,” Science 254:59-67, and Pease et al. (1994)“Light-Generated Oligonucleotide Arrays for Rapid DNA SequenceAnalysis,” Proc. Natl. Acad. Sci. 91:5022-5026.

Nucleic acid segments can also be synthesized by chemical techniques,for example, utilizing the phosphotriester method of Matteucci et al.(1981) J. Am. Chem. Soc. 103:3185. Of course, by chemically synthesizingthe coding sequence, any desired modifications can be made simply bysubstituting the appropriate bases for those encoding the native aminoacid residue sequence. Furthermore, nucleic acid segments are optionallyobtained from existing recombinant DNA molecules (plasmid vectors)containing those genes. Certain of these plasmids are available from theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md., 20852.

The nucleic acid segments and constructs or vectors optionally utilizedin performing the methods of the invention can also be prepared by anumber of other techniques known in the art, such as molecular cloningtechniques. A wide variety of cloning and in vitro amplification methodssuitable for the construction of recombinant nucleic acids, such asexpression vectors, are well-known to persons of skill. Vectors suitablefor use in the present invention are described further below. Generaltexts which describe molecular biological techniques useful herein,including mutagenesis, include Berger and Kimmel, Guide to MolecularCloning Techniques, Methods in Enzymology, Vol. 152, Academic Press,Inc. (1999) (“Berger”); Sambrook et al., Molecular Cloning—A LaboratoryManual 2nd Ed., Vol. 1-3, Cold Spring Harbor Laboratory (2000)(“Sambrook”); and Current Protocols in Molecular Biology, Ausubel et al.(Eds.), Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc., (supplemented through2000) (“Ausubel”). Examples of techniques sufficient to direct personsof skill through in vitro amplification methods, including thepolymerase chain reaction (PCR), the ligase chain reaction (LCR),Qβ-replicase amplification and other RNA polymerase mediated techniques(e.g., NASBA) are found in Berger, Sambrook, and Ausubel, as well asMullis et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide toMethods and Applications (Innis et al., eds.), Academic Press Inc.(1990) (“Innis”); Arnheim & Levinson (1990) Chemical and EngineeringNews 36-47; Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173;Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874; Lomell et al.(1989) J. Clin. Chem. 35:1826; Landegren et al., (1988) Science241:1077-1080; Van Brunt (1990) Biotechnology 8:291-294; Wu and Wallace,(1989) Gene 4:560; Barringer et al. (1990) Gene 89:117, and Sooknananand Malek (1995) Biotechnology 13:563-564. Additional methods of cloningin vitro amplified nucleic acids are also described in U.S. Pat. No.5,426,039 to Wallace et al. Methods of amplifying large nucleic acids byPCR are summarized in Cheng et al. (1994) Nature 369:684-685 and thereferences cited therein, in which PCR amplicons of up to 40 kb aregenerated. One of skill will also appreciate that essentially any RNAcan be converted into a double stranded DNA suitable for restrictiondigestion, PCR expansion and sequencing using reverse transcriptase anda polymerase. See, Ausubel, Sambrook and Berger, all supra.

The isolation of a nucleic acid sequence for inclusion in a vectorconstruct utilized in certain embodiments of the methods of theinvention may be accomplished by any number of techniques known in theart. For instance, oligonucleotide probes based on known sequences canbe used to identify the desired gene in a cDNA or genomic DNA library.Probes may be used to hybridize with genomic DNA or cDNA sequences toisolate homologous genes in the same or different species.Alternatively, antibodies raised against an enzyme can be used to screenan mRNA expression library for the corresponding coding sequence.

Alternatively, the nucleic acids of interest (e.g., genes encodingdesired polypeptides) can be amplified from nucleic acid samples usingamplification techniques. For instance, polymerase chain reaction (PCR)technology can be used to amplify the sequences of desired genesdirectly from genomic DNA, from cDNA, from genomic libraries or cDNAlibraries. PCR and other in vitro amplification methods may also beuseful, for example, to clone nucleic acid sequences that code forproteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of the desired mRNA in samples, for nucleic acidsequencing, or for other purposes. For a general overview of PCR, seeInnis, supra.

Polynucleotides may also be synthesized by well-known techniques asdescribed in the technical literature. See, e.g., Carruthers et al.(1982) Cold Spring Harbor Symp. Quant. Biol. 47:411-418, and Adams etal. (1983) J. Am. Chem. Soc. 105:661. Double stranded DNA segments maythen be obtained either by synthesizing the complementary strand andannealing the strands together under appropriate conditions, or byadding the complementary strand using DNA polymerase with an appropriateprimer sequence.

Oligonucleotides for use as probes, e.g., in in vitro amplificationmethods, for use as gene probes are typically synthesized chemicallyaccording to the solid phase phosphoramidite triester method describedby Beaucage and Caruthers (1981) Tetrahedron Letts. 22(20):1859-1862,e.g., using an automated synthesizer, as described inNeedham-VanDevanter et al. (1984) Nucleic Acids Res. 12:6159-6168.Oligonucleotides for use in the nucleic acid constructs or vectors thatare utilized in certain embodiments of the invention can also be custommade and ordered from a variety of commercial sources known to personsof skill.

VI. Vectors

Essentially any vector or vector system can be used to create thetransformed plants and organogenic cells of the invention. In certainembodiments, nucleic acid segments that encode polypeptides areoperatively linked to vectors in the form of plasmids or plasmid systems(e.g., binary systems, trinary systems, shuttle vector systems, etc.).Certain exemplary plasmid systems that are optionally adapted for use inthe present invention are described in, e.g., U.S. Pat. No. 5,977,439 toHamilton (issued Nov. 2, 1999), U.S. Pat. No. 5,929,306 to Torisky etal. (issued Jul. 27, 1999), U.S. Pat. No. 5,149,645 to Hoekema et al.(issued Sep. 22, 1992), U.S. Pat. No. 6,165,780 to Kawasaki (issued Dec.26, 2000), U.S. Pat. No. 6,147,278 to Rogers et al. (issued Nov. 14,2000), U.S. Pat. No. 4,762,785 to Comai (issued Aug. 9, 1988), and U.S.Pat. No. 5,068,193 to Comai (issued Nov. 26, 1991).

The nucleic acid segments optionally utilized herein, e.g., in the formof expression cassettes typically include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, aDNA sequence of interest (e.g., a gene encoding a polypeptide), and atranscriptional and translational termination region functional in theparticular plant being transformed. The termination region may be nativewith the transcriptional initiation region, may be native with the DNAsequence of interest, or may be derived from another source. Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also, Guerineau et al., (1991), Mol. Gen. Genet., 262:141-144;Proudfoot, (1991), Cell, 64:671-674; Sanfacon et al., (1991), GenesDev., 5:141-149; Mogen et al., (1990), Plant Cell, 2:1261-1272; Munroeet al., (1990), Gene, 91:151-158; Ballas et al., (1989), Nucleic AcidsRes. 17:7891-7903; and Joshi et al., (1987), Nucleic Acid Res.,15:9627-9639).

In certain embodiments, the nucleic acid segments of interest will betargeted to plastids, such as chloroplasts, for expression. In thismanner, where the nucleic acid segment is not directly inserted into theplastid, the expression cassette will additionally contain a geneencoding a transit peptide to direct the nucleic acid of interest to theplastid. Such transit peptides are known in the art. See, e.g., VonHeijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al.(1989) J. Biol. Chem. 264:17544-17550; della-Cioppa et al. (1987) PlantPhysiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res Commun.196:1414-1421; and, Shah et al. (1986) Science 233:478481. Plant genesuseful in the invention may utilize native or heterologous transitpeptides.

Constructs optionally utilized in performing the methods of theinvention may also include any other necessary regulators such as planttranslational consensus sequences (Joshi, C. P., (1987), Nucleic AcidsResearch, 15:6643-6653), introns (Luehrsen and Walbot, (1991), Mol. Gen.Genet., 225:81-93) and the like, operably linked to the nucleotidesequence of interest.

In some embodiments, 5′ leader sequences are included in expressioncassette constructs utilized in performing the methods of the invention.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein et al. (1989) PNAS USA 86:6126-6130); potyvirus leaders,for example, TEV leader (Tobacco Etch Virus) (Allison et al., (1986);MDMV leader (Maize Dwarf Mosaic Virus); Virology, 154:9-20), and humanimmunoglobulin heavy-chain binding protein (BP), (Macejak, D. G., andSarnow, P., (1991), Nature, 353:90-94; untranslated leader from the coatprotein mRNA of alfalfa mosaic virus (AMV RNA 4), (Jobling, S. A., andGehrke, L., (1987), Nature, 325:622-625; tobacco mosaic virus leader(TMV), (Gallie, D. R. et al., (1989), Molecular Biology of RNA, pages237-256; and maize chlorotic mottle virus leader (MCMV) (Lommel, S. A.et al., (1991), Virology, 81:382-385. See also, Della-Cioppa et al.,(1987), Plant Physiology, 84:965-968.

Depending upon where the DNA sequence of interest is to be expressed, itmay be desirable to synthesize the sequence with plant preferred codons,or alternatively with chloroplast preferred codons. Plant preferredcodons may be determined from the codons of highest frequency in theproteins expressed in the largest amount in the particular plant speciesof interest. See, European Patent Application Nos. 0359472 and 0385962;International Application No. WO 91/16432; Perlak et al. (1991) Proc.Natl. Acad. Sci. USA 88:3324-3328; and Murray et al. (1989) NucleicAcids Res. 17: 477-498. In this manner, the nucleotide sequences can beoptimized for expression in plants of interest. It is recognized thatall or any part of the gene sequence may be optimized or synthetic. Thatis, synthetic or partially optimized sequences may also be used. For theconstruction of chloroplast preferred genes, see e.g., U.S. Pat. No.5,545,817.

In preparing expression cassettes, the various nucleic acid fragmentsmay be manipulated, so as to provide for the nucleic acid sequences inthe proper orientation and, as appropriate in the proper reading frame.Towards this end, adapters or linkers may be employed to join thenucleic acid fragments or other manipulations may be involved to providefor convenient restriction sites, removal of superfluous nucleic acidsegments, removal of restriction sites, or the like. For this purpose,in vitro mutagenesis, primer repair, restriction, annealing, resection,ligation, or the like may be employed, where insertions, deletions orsubstitutions, e.g., transitions and transversions, may be involved.

The vectors optionally utilized in performing the methods describedherein may include expression control elements, such as promoters.Polypeptide coding genes are operatively linked to the expression vectorto permit the promoter sequence to direct RNA polymerase binding andexpression of the polypeptide coding gene. Useful in expressing thepolypeptide coding gene are promoters which are inducible, viral,synthetic, constitutive as described in, e.g., Poszkowski et al. (1989)EMBO J. 3:2719 and Odell et al. (1985) Nature 313:810 (1985), andtemporally regulated, spatially regulated, and spatiotemporallyregulated as described in, e.g., Chua et al. (1989) Science 244:174-181.

The choice of which expression vector and, e.g., to which preselectedorgan-enhanced promoter a polypeptide coding gene is operatively linkedtypically depends on the functional properties desired, e.g., thelocation and timing of protein expression. A vector that is useful inpracticing the present invention integrates into the genome of the plantof interest, is capable of directing the replication, and also theexpression of the polypeptide coding gene included in the nucleic acidsegment to which it is operatively linked. It is well known in the artthat the entire expression vector does not integrate into the host plantgenome, but only a portion integrates. Nonetheless, the vector will besaid to integrate for ease of expression.

In some embodiments, a construct utilized in performing the methods ofthe invention may include elements in addition to the conjoined nucleicacid sequences, such as promoters, enhancer elements, and signalingsequences. Exemplary promoters include the CaMV promoter, a promoterfrom the ribulose-1,5-bisphosphate carboxylase-oxygenase small subunitgene, a ubiquitin promoter, and a rolD promoter. Exemplary enhancerelements are described in, e.g., U.S. Pat. No. 6,271,444, which issuedAug. 7, 2001 to McBride et al. Exemplary signaling sequences include,but are not limited to, nucleic acid sequences encoding tissue-specifictransit peptides, such as chloroplast transit peptides (see, e.g., Zhanget al. (2002) Trends Plant Sci 7(1):14-21).

In certain embodiments, a strongly or weakly constitutive plant promotercan be employed which will direct expression of the encoded sequences inall tissues of a plant. Such promoters are active under mostenvironmental conditions and states of development or celldifferentiation. Examples of constitutive promoters include the 1′- or2′-promoter derived from T-DNA of Agrobacterium tumefaciens, and othertranscription initiation regions from various plant genes known to thoseof skill. In situations in which overexpression of a gene isundesirable, a weak constitutive promoters can be used for lower levelsof expression. In instances where high levels of expression are sought,a strong promoter, e.g., a t-RNA or other pol III promoter, or a strongpol II promoter, such as the cauliflower mosaic virus promoter, can beused.

Alternatively, a plant promoter may be under environmental control. Suchpromoters are referred to here as “inducible” promoters. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include pathogen attack, anaerobic conditions, or the presenceof light.

In some embodiments, promoters incorporated into a construct optionallyused to perform the methods of the present invention are“tissue-specific” and, as such, under developmental control in that thedesired gene is expressed only in certain tissues, such asfruit-tissues. In embodiments in which one or more nucleic acidsequences endogenous to the plant are incorporated into a construct, theendogenous promoters (or variants thereof) from these genes can beemployed for directing expression of the genes in the transformed plant.Tissue-specific promoters can also be used to direct expression ofheterologous structural genes, including the artificially evolvednucleic acids described herein.

In addition to the promoters noted above, promoters of bacterial originwhich operate in plants include the octopine synthase promoter, thenopaline synthase promoter and other promoters derived from native Tiplasmids (see, Herrara-Estrella et al. (1983) Nature 303:209-213). Viralpromoters include the 35S and 19S RNA promoters of cauliflower mosaicvirus (Odell et al. (1985) Nature 313:810-812). Other plant promotersinclude the ribulose-1,3-bisphosphate carboxylase small subunit promoterand the phaseolin promoter. The promoter sequence from the E8 gene andother genes may also be used. The isolation and sequence of the E8promoter is described in detail in Deikman and Fischer (1988) EMBO J.7:3315-3327.

In preparing constructs utilized in performing the methods of theinvention, sequences other than the promoter and the conjoined nucleicacid segment can also be employed. If normal polypeptide expression isdesired, a polyadenylation region at the 3′-end of the coding region canbe included. The polyadenylation region can be derived from the naturalgene, from a variety of other plant genes, or from T-DNA.

Typical vectors useful for expression of genes in plants are well knownin the art and include vectors derived from the tumor-inducing (Ti)plasmid of Agrobacterium tumefaciens described by Rogers et al. (1987)Meth. in Enzymol., 153:253 -277 (1987). These vectors are plantintegrating vectors in that on transformation, the vectors integrate aportion of vector DNA into the genome of the plant. For integratingvectors based on the Ti plasmid, the region integrated into the hostplant chromosomes is that between the right and left borders of the Tiplasmid.

Exemplary A. tumefaciens vectors useful herein are plasmids pKYLX6 andpKYLX7 of Schardl et al. (1987) Gene 61:1-11 and Berger et al. (1989)Proc. Natl. Acad. Sci. U.S.A., 86:8402-8406. Plasmid pKYLX6 is an E.coli vector designed for intermediate constructs, whereas plasmid pKYLX7is an A. tumefaciens vector designed for integration of cloned genes.Modified vectors pKYLX61 and pKYLX71 contain HindIII, XhoI, BamHI, PstIand SstI sites in place of the original HindIII-SstI fragment multiplecloning site region. Another useful vector herein is plasmid pBI101.2that is available from Clontech Laboratories, Inc., Palo Alto, Calif.Plasmids pKYLX7, pKYLX71 and pB7101.2 are binary vectors that are usedin A. tumefaciens with another vector having a vir gene. Additionaldetails relating to binary vectors are described in, e.g., Hellens etal. (2000) “pGREEN: a versatile and flexible Ti vector forAgrobacterium-mediated plant transformation,” Plant Molecular Biology42:819-832. Other vectors systems are also optionally utilized hereinincluding, e.g., trinary vector systems. Another plant transformationsystem is based on Agrobacterium rhizogenes that induces hairy rootsrather than a tumor on transformation. See, e.g., InternationalPublication No. WO 88/02405 (published Apr. 7, 1988) describes the useof A. rhizogenes strain A4 and its Ri plasmid along with A. tumefaciensvectors pARC8 or pARC16 to transform plants. Agrobacterium-mediatedtransformation is described further below.

Retroviral expression vectors are also optionally adapted for use inperforming the methods described herein. The term “retroviral expressionvector,” as used herein, refers to a DNA molecule that includes apromoter sequence derived from the long terminal repeat (LTR) region ofa retrovirus genome. Because some of the nucleic acid segment expressionproducts that may be utilized herein are associated with food productionand coloration, the retroviral expression vector is preferablyreplication-incompetent in eukaryotic cells. The construction and use ofretroviral vectors has been described by, e.g., Verma in InternationalPublication No. WO 87/00551, and in Cocking et al. (1987) Science236:1259-62.

In some embodiments, the vector used to express a polypeptide codinggene includes a plant selectable marker that confers a selectablephenotype on the transformed cell. The selectable plant marker gene onthe DNA segment to be inserted will usually encode a function, whichpermits the survival and emergence of transformed organogenic cells ortissue in a selective medium. Usually, the selectable marker gene willencode antibiotic resistance, with suitable genes including those codingfor resistance to the antibiotic spectinomycin (e.g., the aadA gene),the streptomycin phosphotransferase (SPT) gene coding for streptomycinresistance, the neomycin phosphotransferase (NPTII) gene encodingkanamycin or geneticin resistance, the hygromycin phosphotransferase(HPT) gene coding for hygromycin resistance, genes coding for resistanceto herbicides which act to inhibit the action of acetolactate synthase(ALS), in particular the sulfonylurea-type herbicides (e.g., theacetolactate synthase (ALS) gene containing mutations leading to suchresistance in particular the S4 and/or Hra mutations), genes coding forresistance to herbicides which act to inhibit action of glutaminesynthase, such as phosphinothricin or basta (e.g., the bar gene), orother such genes known in the art. The bar gene encodes resistance tothe herbicide basta, the nptII gene encodes resistance to theantibiotics kanamycin and geneticin, and the ALS gene encodes resistanceto the herbicide chlorsulfuron. Selection based on resistance tosulfonylurea-type herbicides is preferred. Selectable markers based onthe green fluorescent protein (GFP) or β-glucuronidase (GUS) are alsooptionally used and are described further in, e.g., Mantis et al. (2000)“Comparing the utility of β-glucuronidase and green fluorescent proteinfor detection of weak promoter activity in Arabidopsis thaliana,” PlantMolecular Biology Reporter 18:319-330.

Methods to select transformed plant cells incorporating a desiredresistance gene are well known in the art. For example, if the marker issulfonylurea resistance, the selection medium generally contains asulfonylurea-type herbicide at an appropriate concentration (e.g.,chlorsulfuron in the range of about 1-1000 μg/l, and more typically inthe range of about 5-100 μg/l). For selection of geneticin resistantplant cells or tissue, which contain the NPTII gene, geneticin istypically included in the medium at 10-50 mg/l. Spectinomycin resistantplant cells or tissue containing the aadA gene are typically selected onmedium containing 200-1000 mg/l spectinomycin.

In certain embodiments, transformed cells and plants are selectedaccording to visual differentiation. For example, since many carotenoidsare colored, these carotenoid products can be visualized and determinedby their characteristic spectra and other analytic methods. Therefore,genes encoding carotenoid biosynthetic enzymes may be used as markergenes to allow for visual selection of transformants. In particular,such transformed cells generally display colors ranging from yellow toorange to red as a result of the increased carotenoid levels. In someembodiments, other analytical techniques can be used to selecttransformed cells including, e.g., mass spectrometry, thin layerchromatography (TLC), high pressure liquid chromatography (HPLC),capillary electrophoresis (CE), NMR spectroscopy, and conventionalhybridization techniques.

VII. Plant Trait Modulation Strategies

The nucleic acid segments introduced into organogenic cells as describedherein may contain one or more genes that are chosen to provide newplant traits, to enhance an existing plant trait, or to otherwise modifyexpression of phenotypes exhibited by the plant. Such traits includeherbicide resistance, pesticide resistance, disease resistance,environmental tolerance (e.g., heat, cold, drought, salinity),morphology, growth characteristics, nutritional content, taste, yield,horticultural characteristics, consumer (quality) traits, and the like.

Functional genes to be introduced may be structural genes, which encodepolypeptides that impart the desired phenotype. Alternatively,functional genes may be regulatory genes that play roles intranscriptional and/or translational control to suppress, enhance, orotherwise modify the transcription and/or expression of endogenous geneswithin the plants. In some embodiments, for example, introduced nucleicacid segments encode polypeptide transcription factors, which whenexpressed in transformed cells effect elevated expression of targetedgenes. In other embodiments, introduced nucleic acid segments encodepromoters and/or enhancers, which nucleic acid segments homologouslyrecombine with promoters and/or enhancers of endogenous genes toincrease or decrease expression of the genes as desired.

To further illustrate, various nucleic acid constructs can be used in anumber of techniques to suppress expression of endogenous plant genes,e.g., sense or antisense suppression or ribozymes. Anti-sense RNAinhibition of gene expression has been shown; see, e.g., Sheehy et al.(1988) Proc. Nat. Acad. Sci. USA 85:8805-8809, and Hiatt et al. U.S.Pat. No. 4,801,340. For examples of the use of sense suppression tomodulate expression of endogenous genes see, Napoli et al. (1990) ThePlant Cell 2:279-289, and U.S. Pat. No. 5,034,323.

Catalytic RNA molecules or ribozymes can also be used to inhibit geneexpression, which is optionally used to effect the accumulation ofselected products that are upstream in a given biochemical pathway fromthe gene that is inhibited. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA-cleaving activity upon them,thereby increasing the activity of the constructs.

A number of classes of ribozymes have been identified, which areoptionally adapted for use in performing the methods described herein.One class of ribozymes is derived from a number of small circular RNAswhich are capable of self-cleavage and replication in plants. The RNAsreplicate either alone (viroid RNAs) or with a helper virus (satelliteRNAs). Examples include RNAs from avocado sunblotch viroid and thesatellite RNAs from tobacco ringspot virus, lucerne transient streakvirus, velvet tobacco mottle virus, solanum nodiflorum mottle virus, andsubterranean clover mottle virus. The design and use of targetRNA-specific ribozymes is described in Haseloff et al. (1988) Nature334:585-591.

For antisense suppression, the introduced sequence also need not be fulllength relative to either the primary transcription product or fullyprocessed mRNA. Generally, higher homology can be used to compensate forthe use of a shorter sequence. Furthermore, the introduced sequence neednot have the same intron or exon pattern, and homology of non-codingsegments may be equally effective. Normally, a sequence of between about30 or 40 nucleotides and about 2000 nucleotides should be used, though asequence of at least about 100 nucleotides is preferred, a sequence ofat least about 200 nucleotides is more preferred, and a sequence of atleast about 500 nucleotides is especially preferred.

To further illustrate, RNA interference (RNAi), also known asPost-Transcriptional Gene Silencing (PTGS), is also optionally utilizedto modulate traits in plants. RNAi is a cellular mechanism thatselectively negates the effect of a target gene by destroying messengerRNA. By destroying the targeted mRNA, protein synthesis is interrupted,thereby effectively “silencing” the target gene. In certain embodiments,this process is initiated by double-stranded RNA (dsRNA), where onestrand is substantially identical to the target mRNA sequence.Accordingly, in some embodiments of the invention, nucleic acid segmentsintroduced into plant cells as described herein trigger the productionof double stranded dsRNA, which is then cleaved into small interferingRNA (siRNA) as part of the RNAi process. This results in the destructionof the target mRNA, thereby effectively silencing expression of thetarget gene. Additional details relating to RNAi are described in, e.g.,U.S. Pat. No. 6,573,099, entitled “GENETIC CONSTRUCTS FOR DELAYING ORREPRESSING THE EXPRESSION OF A TARGET GENE,” which issued Jun. 3, 2003to Graham, and in, e.g., Arenz et al. (2003) “RNA interference: from anancient mechanism to a state of the art therapeutic application?”Naturwissenschaften. 90(8):345-59, Wang et al. (2003) “RNA interference:antiviral weapon and beyond,” World J Gastroenterol. 9(8):1657-61, andLavery et al. (2003) “Antisense and RNAi: powerful tools in drug targetdiscovery and validation” Curr Opin Drug Discov Devel. 6(4):561-9.Custom nucleic acid segments that can be utilized to effect target genesilencing are also commercially available from various suppliers, suchas Ambion, Inc. (Austin, Tex., USA), Benitec Australia Limited (StLucia, AU), and the like.

Often the functional genes to be introduced will be modified from theirnative form. For example, sense and anti-sense constructs referred toabove often have all or a portion of the transcript of the native geneoperably linked to a promoter sequence at the 5′ end of thetranscribable segment, and operably linked to the 3′ sequence of anothergene (including polyadenylation sequences) at the 3′ end of thetranscribable segment. As is apparent to those skilled in the art, thepromoter sequence could be one of the many plant active sequencesalready described. Alternatively, other plant-active promoter sequencescan be derived specifically to be linked to the transcribable segment.The promoter can be endogenous to pineapple, or can be from an exogenoussource such as a cauliflower mosaic virus 35S promoter (Odell et al.(1985) Nature 313:810-812), the ubiquitin 1 promoter (Christiensen etal. (1992) Plant Mol. Biol. 18:675-689), or the Smas promoter (Ni et al.(1995) Plant J. 7:661-676). The 3′ end sequence to be added can bederived from, preferably, the nopaline synthase or octopine synthasegenes, or alternatively from another plant gene, or less preferably fromany other eukaryotic gene.

As described herein, the production of carotenoids can be elevated incells and plants transformed with nucleic acid segments (e.g., a firstgene of interest) that, e.g., encode carotenoid biosynthetic enzymes.Optionally, once this biosynthetic activity has been increased byexpression of these introduced carotenoid biosynthesis genes, thepathway can be diverted for the production and accumulation of specificcarotenoids. The diversion typically includes the use of at least onesecond gene of interest. To illustrate, the second gene can encode anenzyme to force the production of a particular carotenoid oralternatively can encode a gene to stop the pathway for the accumulationof a particular carotenoid. To force the production of a particularcarotenoid, expression of a carotenoid biosynthesis gene in the pathwayfor the desired carotenoid is used. Genes native or exogenous to thetarget plant are optionally used in these methods, including, e.g.,carotenoid biosynthesis genes from sources other than plants, such asbacteria, including Erwinia and Rhodobacter species. Exemplarycarotenoid biosynthesis genes that can be utilized for these purposesare described further above. To stop the pathway in order to accumulatea particular carotenoid compound, the second gene will provide forinhibition of transcription of a gene (e.g., native or exogenous) to thetarget plant in which the enzyme encoded by the inhibited gene iscapable of modifying the desired carotenoid compound. Inhibition may beachieved by transcription of the gene to be inhibited in either thesense (cosuppression) or antisense orientation of the gene. Other senseand antisense strategies for modulating carotenoid accumulation inplants are referred to above.

To further illustrate, but not to limit the present invention, to alter,e.g., the carotenoid composition of a plant towards the accumulation ofhigher levels of β-carotene derived carotenoids, such as zeaxanthin,zeaxanthin diglucoside, canthaxanthin, and astaxanthin, inhibition oflycopene ε-cyclase can be achieved to prevent accumulation of α-caroteneand other carotenoids that are derivative from α-carotene, such aslutein. In addition to the inhibition of lycopene ε-cyclase, increasedexpression of a second gene may be utilized for increased accumulationof a particular β-carotene derived carotenoid. For example, increasedβ-carotene hydroxylase expression is useful for production ofzeaxanthin, whereas increased β-carotene hydroxylase andketo-introducing enzyme expression is useful for production ofastaxanthin. Alternatively, to accumulate lycopene, the inhibition oflycopene β-cyclase or of lycopene ε-cyclase and lycopene β-cyclase canbe effected to reduce conversion of lycopene to α- and β-carotene.

A variety of genes are optionally used as to divert carotenoidbiosynthesis in cells and plants as desired. These include, but are notlimited to, β-carotene hydroxylase or crtZ (Hundle et al. (1993) FEBSLett. 315:329-334, Accession No. M87280) for the production ofzeaxanthin; genes encoding keto-introducing enzymes, such ascrtW (Misawaet al. (1995) J. Bacteriol. 177:6575-6584, WO 95/18220, WO 96/06172) orβ-C-4-oxygenzse (crtO; Harker et al. (1997) FEBS Lett. 404:129-134) forthe production of canthaxanthin; crtZ and crtW or crtO for theproduction of astaxanthin; ε-cyclase and ε-hydroxylase for theproduction of lutein; ε-hydroxylase and crtZ for the production oflutein and zeaxanthin; antisense lycopene ε-cyclase (Accession No.U50738) for increased production of β-carotene; antisense lycopeneε-cyclase and lycopene β-cyclase (Hugueney et al. (1995) Plant J.8:417-424, Cunningham Jr et al. (1996) Plant Cell 8:1613-1626, Scolniket al. (1995) Plant Physiol. 108:1343, Accession Nos. X86452, L40176,X81787, U50739 and X74599) for the production of lycopene; antisenseplant phytoene desaturase for the production of phytoene; and the like.

In this manner, the pathway can be modified for the high production ofany particular carotenoid compound of interest. Such compounds include,but are not limited to, α-cryptoxanthin, γ-cryptoxanthin, ζ-carotene,phytofluene, neurosporane, etc. Using the methods of the invention, anycompound of interest in the carotenoid pathway can be produced at highlevels in selected storage organs, such as the fruit of plants.

Optionally, the pathway can also be manipulated to decrease levels of,for example, a particular carotenoid by, e.g., transforming the plantcell with antisense DNA sequences which prevents the conversion of theprecursor compound into the particular carotenoid being regulated.

Although some of the description herein relates to altering theaccumulation of carotenoids in plants for purposes of clarity ofillustration, it will be appreciated that the general strategiesdescribed herein can be readily adapted to modulate other plant traitsby persons skilled in the art. Accordingly, the present invention is notlimited to modulated carotenoid accumulation in plants.

VIII. Examples

The following examples are offered to illustrate, but not to limit theclaimed invention.

Media are designated with letters and numbers; letters refer to mediacomponents used, followed by a number that indicates the concentrationof the particular component. For example, B2N2 is an MS media thatcontains 6-benzylamino purine (BA or B) and α-naphthalene acetic acid(NAA or N). More specific details relating to the media compositionsreferred to in these examples, including component concentrations, areprovided below.

Example 1 Transformation and Regeneration of Pineapple Del Monte GoldMD-2 Using Agrobacterium tumefaciens Strain C58PMP90/PDM

1. Preparation of Source Tissues (Transgenic Line 2.48.5.1)

i. Establishment of Shoot Cultures.

Meristems isolated from crowns of Del Monte Gold MD-2 pineapples grownin the field in Costa Rica were used to establish shoot cultures.Briefly, leaves of the crowns were removed by hand and discarded, thecore or the stem of the crown (˜3×5 cm) was washed in water, surfacesterilized with 33% Clorox plus 0.05% Tween20 with stirring for 20minutes and rinsed twice in sterile water. The lateral meristems andcrown tip meristem were excised from the core by removing primary leavesone by one, while flaming the tools frequently. The crown tip meristemexplant including the meristem dome, 2-3 tiny primary leaves, and 1 cm³of the stem core was placed on the shoot culture medium B2N2+CC andafter 9 days to B2N2+N and after another 8 days to liquid B3 medium.Additional details relating to culture media are provided below. Thelateral meristems were also isolated along with 1 cm³ of the stem coreand cultured on the same media as above. Cultures were incubated undercontinuous light at 28° C. After 20 days from initial culture, crown tipleaves had grown long, these were removed to promote the growth of crowntip meristem and were transferred to fresh B2N2 medium. After a total of4 months from culture initiation, shoot clusters were transferred andmaintained in liquid B1.5N.5 medium with monthly subculture.

2. Pretreatment of Explants for Cocultivation with Agrobacterium.

Rapidly growing shoots were used as explants. Tips of the long leaves(>15 mm) were cut off to provide smaller explants. Shoots were cutlongitudinally into 4 sections, then leaf bases, core sections andlongitudinal sections were pretreated (cultured) on P10T2.2A for 30 daysthen additional leaf bases and core sections were prepared and culturedon same medium until morphogenic tissues were produced. The tissues weresubcultured on P10 medium for 5 weeks, then mixed with Agrobacterium forcocultivation.

3. Agrobacterium tumefaciens Culture and Preparation.

Agrobacterium tumefaciens strain C58pmp90 containing a vector systemcontaining a binary vector system was used for transformation. Thevector system included vector pDM containing surB gene, which conferschlorsulfuron resistance, and the gus gene encoding beta-glucuronidase.Bacteria were taken out of frozen glycerol, cultured and maintained onL-Broth medium solidified with 1.5% Bactoagar containing 10 mg/ltetracycline. One day before cocultivation, bacteria were scraped offthe solid medium using a loop and suspended inn liquid MinAsuc mediumand cultured for one day on a shaker (120 rpm) at 28° C. Bacteriaconcentration was determined, using a spectrophotometer (Beckman DU-50)before cocultivation, and was shown to be 8×10⁸ cells/ml.

4. Cocultivation on Cocultivation Medium.

Bacteria were mixed at the volume ratio of 3:1 (plant tissue:Agrobacterium cells) with 80 leaf bases and core sections, tissues wereblotted dry and the mixture was placed on 7.0 cm sterile Fisherbrand G6glass filter circles on the top of cocultivation medium P10As100. Plateswere parafilmed and placed in a 24° C. controlled environment incubatorin the dark for 3 days.

5. Recovery, Selection and Plant Regeneration.

After cocultivation, tissues were transferred (˜20 pieces/plate) torecovery medium P10Vanc100 for a recovery period of 11 days under lowlight condition (16 hrs/day). The explants then were transferred toselection medium P10Carb300CS10 for 25 days, P10Carb300CS20 for 28 days,T1.1I.1Carb250CS20 for 28 days, B3N.2Carb200CS10 for 28 days,B3N.2Cef400CS10 for 28 days. Carbenicillin or cefotaxime was used tokill off the residual Agrobacterium and chlorsulfuron (CS) to select fortransformed cells. On selection medium, most of the tissues turned brownwithin 3-6 weeks, however occasionally some sectors of the tissuesremained healthy and started growing to produce green healthyorganogenic tissue. Transgenic shoots were subcultured ontoB3N.2Carb100CS10 for 30 days to produce multiple shoots and shoot buds.Some of the shoots were vitrified, which then were transferred to B1A1%Carb100CS10 for 14 days to produce elongated normal shoots.

6. Confirmation of Transformation.

Shoots were confirmed to be transformed by different means: 1. CSresistant shoots or shoot primordia remained healthy and green on lethallevels of CS, 2. CS resistant shoots or shoot primordia were sampled(2-5 mg/resistant piece) for GUS assay. Transformed tissues stained blueand nontransformed tissues did not stain blue. Tissues can also betested molecularly for transformation using PCR and, e.g., Southernblotting analyses.

7. Micropropagation and Production of Transgenic Plants.

Individual or small shoot clusters were transferred to 15 ml liquidmedium B1Carb100CS20 in GA-7 cubes (2-3 clusters/cube) for propagationand growth. Optionally, 30 days later, shoot clusters can bemicropropagated and maintained in B1.5N.5CS20 (containing nocounterselective agents) for several months. The individual shoots (4-6cm long) were separated and cultured in liquid rooting mediumN.5IBA.5CS10 or N.5IBA.5CS20 (containing no counterselective agents) for2-4 weeks to produce complete plants. Plants are transplanted in soil,hardened off gradually and then transferred to the greenhouseconditions.

Example 2 Transformation and Regeneration of Pineapple Del Monte GoldMD-2 Using Agrobacterium tumefaciens Strain C58PMP90/PDM

1. Preparation of Source Tissues (Transgenic Line 16.5.4)

i. Establishment of Shoot Cultures.

Meristems isolated from crowns of Del Monte Gold MD-2 pineapples grownin the field in Costa Rica were used to establish shoot cultures.Briefly, leaves of the crowns were removed by hand and discarded, thecore or the stem of the crown (˜3×5 cm) was washed in water, surfacesterilized with 33% Clorox plus 0.05% Tween20 with stirring for 25minutes and rinsed twice in sterile water. The lateral meristems andcrown tip meristem were excised from the core by removing primary leavesone by one, while flaming the tools frequently. The crown tip meristemexplant including the meristem dome, 2-3 tiny primary leaves, and 1 cm³of the stem core was placed on the shoot culture medium B2N2. Thelateral meristems were also isolated along with 1 cm³ of the stem coreand cultured on the same medium. Cultures were incubated undercontinuous light at 28° C. Nine days after culture initiation, thetissues were subcultured onto B2N2. After 20 days, crown tip leaves hadgrown long, these were removed to promote the growth of crown tipmeristem and were transferred to fresh B2N2 medium. After two additionalweeks, buds were formed (2-3 per explant) and subsequently new smallshoots were produced to form a cluster of shoots. After a total of 4months from culture initiation, shoot clusters were transferred andmaintained in liquid B1.5N.5 medium with monthly subculture.

2. Pretreatment of Explants for Cocultivation with Agrobacterium.

Rapidly growing shoots were used as explants. Tips of the long leaves(>15 mm) were cut off to provide smaller explants. Shoots were cutlongitudinally into 4-6 sections and pretreated (cultured) on P10T1.1b6(containing 6% banana pulp) and P2B.5 for 8 days. Leaf bases and coresections were prepared and were mixed with Agrobacterium forcocultivation.

3. Agrobacterium tumefaciens Culture and Preparation.

Agrobacterium tumefaciens strain C58 pmp90 containing the binary vectorsystem, referred to above, was used for transformation. Morespecifically, the vector system included vector pDM containing a surBgene and a gus gene. Bacteria were taken out of frozen glycerol,cultured and maintained on L-Broth medium solidified with 1.5% Bactoagarcontaining 10 mg/l tetracycline. One day before cocultivation, bacteriawere scraped off the solid medium using a loop and suspended in liquidMinAsuc medium and cultured for one day on a shaker (120 rpm) at 28° C.Bacteria concentration was determined, using a spectrophotometer(Beckman DU-50) before cocultivation, and was shown to be 4.4×10⁸cells/ml.

4. Cocultivation on Cocultivation Medium.

Bacteria were mixed at the volume ratio of 4:1 (plant tissue:Agrobacterium cell) with 24 leaf bases and core sections, tissues wereblotted dry and the mixture was placed on 7.0 cm sterile Fisherbrand G6glass filter circles on the top of cocultivation medium P10T2.2As300.Plates were parafilmed and placed in a 24° C. controlled environmentincubator in the dark for 3 days.

5. Recovery and Selection.

After cocultivation, tissues were transferred (15-20 pieces/plate) torecovery medium P10T1.1Carb500 for a recovery period of 7 days under lowlight condition (16 hrs/day). The explants then were transferred toselection medium P10T2.2Carb300CS5 for 27 days. Carbenicillin was usedto kill off the residual Agrobacterium and chlorsulfuron (CS) to selectfor transformed cells. On selection medium, most of the tissues turnedbrown within 3-6 weeks, however occasionally some sectors of the tissuesremained healthy and started growing to produce green healthymorphogenic tissue.

6. Confirmation of Putative Transformed Shoots.

Same as Example 1.

7. Micropropagation and Production of Transgenic Plants.

Same as Example 1.

Example 3 Transformation and Regeneration of Pineapple Del Monte GoldMD-2 Using Agrobacterium tumefaciens Strain C58PMP90/PDM (TransgenicLine 16.1.5, 16.1.6, and 16.1.7)

1. Preparation of Source Tissues

i. Establishment of Shoot Cultures.

Same as Example 2.

2. Pretreatment of Explants for Cocultivation with Agrobacterium.

Rapidly growing shoots were used as explants. Tips of the long leaves(>15 mm) were cut off to provide smaller explants. Shoots were cutlongitudinally into 3-7 sections and pretreated (cultured) on B3N.2medium for 12 days to produce organogenic cells and tissues, thenmaintained on B3N.2 medium with subculturing schedule of 28 and 31 days,then cut into 2-4 mm sections and pretreated on B3N.2 medium for 7 days.The sections were mixed with Agrobacterium for cocultivation.

3. Agrobacterium tumefaciens Culture and Preparation.

Agrobacterium tumefaciens strain C58 pmp90 containing the binary vectorsystem, referred to above, was used for transformation. The vectorsystem included vector pDM containing a surB gene and a gus gene.Bacteria were taken out of frozen glycerol, cultured and maintained onL-Broth medium solidified with 1.5% Bactoagar containing 10 mg/ltetracycline. One day before cocultivation, bacteria were scraped offthe solid medium using a loop and suspended in liquid MinAsuc medium andcultured for one day on a shaker (120 rpm) at 28° C. Bacteriaconcentration was determined, using a spectrophotometer (Beckman DU-50)before cocultivation, and was shown to be 2, 5 and 10×10⁸ cells/ml.

4. Cocultivation on Cocultivation Medium.

Bacteria were mixed at the volume ratio of 4:1 (plant tissue:Agrobacterium cell) with 24 leaf bases and core sections, tissues wereblotted dry and the mixture was placed on 7.0 cm sterile Fisherbrand G6glass filter circles on the top of cocultivation medium B3N.2As300 orB3N.2As1000. Plates were parafilmed and placed in a 24° C. controlledenvironment incubator in the dark for 3 days.

5. Recovery and Selection.

After cocultivation, tissues were transferred (˜15 pieces/plate) torecovery medium B3N.2Carb500 for a recovery period of 6 or 7 days underlow light condition (16 hrs/day). The explants then were transferred toselection medium B3N.2Carb300CS5 for 27 days. Carbenicillin was used tokill off the residual Agrobacterium and chlorsulfuron (CS) to select fortransformed cells. On selection medium, most of the tissues turned brownwithin 3-6 weeks, however, occasionally some sectors of the tissuesremained healthy and started growing to produce green healthymorphogenic tissue.

6. Confirmation of Putative Transformed Shoots.

Same as Example 1.

7. Micropropagation and Production of Transgenic Plants.

Same as Example 1.

Example 4 Transformation and Regeneration of Pineapple Del Monte GoldMD-2 Using Agrobacterium tumefaciens Strain C58PMP90/PDM (TransgenicLine 15.56)

1. Preparation of Source Tissues

i. Establishment of Shoot Cultures.

Same as Example 1.

2. Pretreatment of Explants for Cocultivation with Agrobacterium.

Rapidly growing shoots were used as explants. Tips of the long leaves(>15 mm) were cut off to provide smaller explants. Shoots were cutlongitudinally into 3-7 sections and pretreated (cultured) on Dic2.5B.5medium for 8 days, then leaf bases and longitudinal sections wereprepared and mixed with Agrobacterium for cocultivation.

3. Agrobacterium tumefaciens Culture and Preparation.

Agrobacterium tumefaciens strain C58 pmp90 containing the binary vectorsystem, referred to above, was used for transformation. As mentioned,the vector system included vector pDM containing a surB gene and a gusgene. Bacteria were taken out of frozen glycerol, cultured andmaintained on L-Broth medium solidified with 1.5% Bactoagar containing10 mg/l tetracycline. One day before cocultivation, bacteria werescraped off the solid medium using a loop and suspended in liquidMinAsuc medium and cultured for one day on a shaker (120 rpm) at 28° C.Bacteria concentration was determined, using a spectrophotometer(Beckman DU-50) before cocultivation, and was shown to be 3×10⁸cells/ml.

4. Cocultivation on Cocultivation Medium.

Bacteria were mixed at the volume ratio of 3:1 (plant tissue:Agrobacterium cell) with 24 leaf bases and core sections, tissues wereblotted dry and the mixture was placed on 7.0 cm sterile Fisherbrand G6glass filter circles on the top of cocultivation medium Dic2.5B.5As300.Plates were parafilmed and placed in a 24° C. controlled environmentincubator in the dark for 3 days.

5. Recovery and Selection.

After cocultivation, tissues were transferred (˜15 pieces/plate) torecovery medium Dic2.5B.5Carb500 for a recovery period of 10 days underlow light condition (16 hrs/day). The explants then were transferred toselection medium Dic2.5B.5Carb500CS5 for 31 days with monthly subculturethereafter. Carbenicillin was used to kill off the residualAgrobacterium and chlorsulfuron (CS) to select for transformed cells. Onselection medium, most of the tissues turned brown within 3-6 weeks,however, occasionally some sectors of the tissues remained healthy andstarted growing to produce green healthy morphogenic tissue.

6. Confirmation of Putative Transformed Shoots.

Same as Example 1.

7. Micropropagation and Production of Transgenic Plants.

Same as Example 1.

Example 5 Transformation and Regeneration of Pineapple Del Monte GoldMD-2 USING Agrobacterium tumefaciens Strain C58PMP90/PDM (TransgenicLine 2.77.1.1)

1. Preparation of Source Tissues

i. Establishment of Shoot Cultures.

Similar to Example 1 with some changes. Meristems were isolated fromcrowns of Del Monte Gold MD-2 pineapples grown in the field in Hawaiiwere used to establish shoot cultures. Briefly, leaves of the crownswere removed by hand and discarded, the core or the stem of the crown(˜3×5 cm) was washed in water containing liquid soap, surface sterilizedwith 70% ethanol for 1 minute, 25% Clorox plus 0.05% Tween20 withstirring for 25 minutes and rinsed twice in sterile water. The lateralmeristems and crown tip meristem were excised from the core by removingprimary leaves one by one, while flaming the tools frequently. The crowntip meristem explant including the meristem dome, 2-3 tiny primaryleaves, and 1 cm³ of the stem core was placed on the shoot culturemedium B2N2. After 8 days the buds had grown and the explants weredivided into smaller pieces and transferred to fresh B2N2 medium, and 10days later to B3 medium. After additional 5 weeks the shoots werecultured in liquid B1.5N.5 medium with monthly subculture.

2. Pretreatment of Explants for Cocultivation with Agrobacterium.

No pretreatment was performed in this example. Rapidly growing shootswere used as explants. Tips of the long leaves (>15 mm) were cut off toprovide smaller explants. Leaf bases and core sections were prepared andwithout pretreatment were mixed with Agrobacterium for cocultivation.

3. Agrobacterium tumefaciens Culture and Preparation.

Same as Example 1. Bacteria concentration was shown to be 6×10⁸cells/ml.

4. Cocultivation on Cocultivation Medium.

Bacteria were mixed at the volume ratio of 3:1 (plant tissue:Agrobacterium cell) with 80 leaf bases and core sections, tissues wereblotted dry and the mixture was placed on 7.0 cm sterile Fisherbrand G6glass filter circles on the top of cocultivation medium P10T2.2AAs300.Plates were parafilmed and placed in a 24° C. controlled environmentincubator in the dark for 3 days.

5. Recovery and Selection.

After cocultivation, tissues were transferred (15-18 pieces/plate) torecovery medium P10T2.2Carb300 for a recovery period of 8 days under lowlight condition (16 hrs/day). The explants then were transferred toselection medium P10T2.2Carb250CS10 for 21 days P10T2.2Carb200CS10 for28 days, P10T2.2Carb300CS5 for 20 days, P10T2.2Carb300CS5 for 30 days,B3N.2Carb100CS10 for 25 days. Carbenicillin was used to kill off theresidual Agrobacterium and chlorsulfuron (CS) to select for transformedcells. On selection medium, most of the tissues turned brown within 3-4weeks, however, occasionally some sectors of the tissues remainedhealthy and started growing to produce green healthy organogenic tissue.The organogenic tissues produced shoot on B3N.2 and B1 media series

6. Confirmation of Putative Transformed Shoots.

Same as Example 1.

7. Micropropagation and Production of Transgenic Plants.

Same as Example 1.

Example 6 Transformation and Regeneration of Pineapple Del Monte GoldMD-2 Using Agrobacterium tumefaciens strain C58PMP90/PDM (TransgenicLine 13.12.1, 13.12.2, 13.12.4)

1. Preparation of Source Tissues

i. Establishment of Shoot Cultures.

Similar to Example 5.

2. Preparation of Explants for Cocultivation with Agrobacterium.

No pretreatment was performed in this example. Rapidly growing shootswere used as explants. Tips of the long leaves (>15 mm) were cut off toprovide smaller explants. Leaf bases and core sections were prepared andwithout pretreatment were mixed with Agrobacterium for cocultivation.

3. Agrobacterium tumefaciens Culture and Preparation.

Same as Example 1. Two bacteria samples were used and the concentrationswere shown to be 7×10⁸ cells/ml and 9×10⁸ cells/ml.

4. Cocultivation on Cocultivation Medium.

Bacteria were mixed at the volume ratio of 3:1 (plant tissue:Agrobacterium cell) with ˜50 leaf bases and core sections for eachtreatment, the mixtures were vacuum infiltrated, then the tissues wereblotted dry (13.12.1) or not (13.12.2 and 13.12.4) and the mixture wasplaced on 7.0 cm sterile Fisherbrand G6 glass filter circles on the topof cocultivation medium P10T2.2AAs300. Plates were parafilmed and placedin a 24° C. controlled environment incubator in the dark for 3 days.

5. Recovery and Selection.

After cocultivation, tissues were transferred (15-18 pieces/plate) torecovery medium P10T2.2ACarb300 for a recovery period of 6 days underlow light condition (16 hrs/day). The explants with morphogenicpotentials were transferred to selection medium P10T2.2Carb300CS5 for21-24 days for three rounds of selection, then on B3N.2Carb100Cs10 forselection and shoot production. On selection medium, most of the tissuesturned brown within 2-3 weeks, however, some sectors of the tissuesremained healthy and started growing to produce green healthyorganogenic tissue. The organogenic tissues produced shoot on B3N.2 andB1 media series.

6. Confirmation of Putative Transformed Shoots.

Same as Example 1.

7. Micropropagation and Production of Transgenic Plants.

Same as Example 1.

Media Compositions

Minimal Asuc = MinAsuc preferred range potassium phosphate dibasic 10.5g/l 5-20 g/l potassium phosphate monobasic 4.5 g/l 2-8 g/l ammoniumsulfate 1.0 g/l 0.5-3 g/l sodium citrate dihydrate 0.5 g/l 0-2 g/lmagnesium sulfate heptahydrate 247 mg/l 0-1000 g/l glucose 2.0 g/l 1-30g/lMinAsucMinA but with sucrose instead of glucose.L-Broth:

Tryptone 10 g/l  Yeast Extract 5 g/l NaCl 5 g/l Glucose 1 g/l pH 7.0-7.2Bacto Agar 15 g/l 

For tissue culture media, the pH should be between about 5-7.5,preferably about 5.6. The medium is used following sterilization byautoclaving, except for specific components that are filter-sterilizedand then added after autoclaving.

MS MS salts 1X B5 vitamins 1X Sucrose 30 g/l MES 600 mg/l Gel-rite ® 2.5g/l pH 5.7 B2N2 MS medium + BA 2 mg/l NAA 2 mg/l B2N2 + CC B2N2 +Carbenicillin 500 mg/l Cefotaxime 500 mg/l B2N2 + N B2N2 + Nystatin 40mg/l B1.5N.5 LIQUID MS without Gel-rite + BA 1.5 mg/l NAA 0.5 mg/lB1CS10 MS + BA 1 mg/l Chlorsulfuron 10 μg/l B1CARB100CS10 B1CS10 +Carbenicillin 100 mg/l B3 (LIQUID) MS without Gel-rite + BA 3 mg/l B3N.2MS + BA 3 mg/l NAA 0.2 mg/l B3N.2AS300 B3N.2 + Acetosyringone 300 μMB3N.2AS1000 B3N.2 + Acetosyringone 1000 μM B3N.2CARB500 B3N.2 +Carbenicillin 500 mg/l B3N.2CARB100CS10 B3N.2 + Carbenicillin 100 mg/lChlorsulfuron 10 μg/l B3N.2CARB300CS5 B3N.2 + Carbenicillin 300 mg/lChlorsulfuron 5 μg/l B3N.2CARB200CS10 B3N.2 + Carbenicillin 200 mg/lChlorsulfuron 10 μg/l B3N.2CEF400CS10 B3N.2 + Cefotaxime 400 mg/lChlorsulfuron 10 μg/l B3N.2CS20 B3N.2 + Chlorsulfuron 20 μg/l B1 MS + BA1 mg/l B1A1% B1 + Agar 10 g/l (instead of Gel-rite) B1 LIQUID B1 withoutGel-rite B3 MS + BA 3 mg/l DIC2.5B.5 MS + Dicamba 2.5 mg/l BA 0.5 mg/lDIC2.5B.5AS300 Dic2.5B.5 + Acetosyringone 300 μM DIC2.5B.5CARB500Dic2.5B.5 + Carbenicillin 500 mg/l DIC2.5B.5CARB500CS5Dic2.5B.5Carb500 + Chlorsulfuron 5 μg/l P2B.5 MS + Picloram 2 mg/l BA0.5 mg/l P10 MS + Picloram 10 mg/l P10AS100 P10 + Acetosyringone 100 μMP10CARB300CS10 P10 + Carbenicillin 300 mg/l Chlorsulfuron 10 μg/lP10CARB300CS20 P10Carb300 + Chlorsulfuron 20 μg/l P10T1.1B6 MS +Picloram 10 mg/l Thidiazuron 1.1 mg/l Banana Pulp 6% P10T1.1CARB500 MS +Picloram 10 mg/l Thidiazuron 1.1 mg/l Carbenicillin 500 mg/l P10T2.2MS + Picloram 10 mg/l Thidiazuron 2.2 mg/l P10T2.2A P10T2.2 + agarinstead of gel-rite P10T2.2AAS300 P10T2.2A + Acetosyringone 300 μMP10T2.2ACARB 300 P10T2.2A + Carbenicillin 300 mg/l P10T2.2AS300P10T2.2 + Acetosyringone 300 μM P10T2.2CARB200CS10 P10T2.2 +Carbenicillin 200 mg/l Chlorsulfuron 10 μg/l P10T2.2CARB250CS10P10T2.2 + Carbenicillin 250 mg/l Chlorsulfuron 10 μg/l P10T2.2CARB300P10T2.2 + Carbenicillin 300 mg/l 300 P10T2.2CARB300CS5 P10T2.2Carb300 +Chlorsulfuron 5 μg/l P10B.5 MS + Picloram 10 mg/l BA 0.5 mg/l P10VANC100MS + Picloram 10 mg/l Vanc 100 mg/l N.5I.5 LIQUID MS without Gel-rite +NAA 0.5 mg/l IBA 0.5 mg/l T1.1I.1CARB250CS20 MS + Thidiazuron 1.1 mg/lIBA 0.1 mg/l Carbenicillin 250 mg/l Chlorsulfuron 20 μg/l

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, all the techniques and apparatus described abovecan be used in various combinations. All publications, patents, patentapplications, and/or other documents cited in this application areincorporated by reference in their entirety for all purposes to the sameextent as if each individual publication, patent, patent application,and/or other document were individually indicated to be incorporated byreference for all purposes.

1. A method of generating a plant comprising transformed plant cells,the method comprising: culturing at least one non-apical meristemic cellto produce at least one shoot; culturing at least one leaf base explantfrom the shoot to produce one or more organogenic cells; introducing atleast one nucleic acid segment into the organogenic cells to produce oneor more transformed organogenic cells, wherein the nucleic acid segmentis introduced into the organogenic cells using Agrobacterium-mediateddelivery; and generating at least one plant from the transformedorganogenic cells without going through an undifferentiatedintermediate.
 2. The method of claim 1, wherein the non-apicalmeristemic cell comprises a pineapple cell.
 3. The method of claim 1,wherein the nucleic acid segment comprises at least one sense nucleicacid segment that corresponds to at least a portion of at least oneendogenous gene; wherein the nucleic acid segment comprises at least onesense nucleic acid segment that corresponds to at least a portion of atleast one exogenous gene; wherein the nucleic acid segment comprises atleast one antisense nucleic acid segment that corresponds to at least aportion of at least one endogenous gene; wherein the nucleic acidsegment encodes at least one polypeptide transcription factor; or,wherein the nucleic acid segment encodes at least one promoter and/or atleast one enhancer, which nucleic acid segment homologously recombineswith at least one promoter and/or at least one enhancer of at least oneendogenous gene.
 4. The method of claim 1, wherein the nucleic acidsegment encodes a polypeptide.
 5. The method of claim 4, wherein thepolypeptide is heterologous to the organogenic cells.
 6. The method ofclaim 4, wherein the polypeptide is homologous to at least oneendogenous polypeptide of the organogenic cells.
 7. The method of claim4, wherein the polypeptide comprises at least one carotenoidbiosynthetic polypeptide that is selected from the group consisting of:an isomerase, an isopentenyl diphosphate isomerase, a geranylgeranylpyrophosphate synthase, a phytoene synthase, a phytoene desaturase, aζ-carotene desaturase, a lycopene β-cyclase, a lycopene ε-cyclase, aβ-carotene hydroxylase, and an ε-hydroxylase.